Compositions and methods for epigenome editing

ABSTRACT

Disclosed herein are CRISPR/Cas9-based gene activation systems that include a fusion protein of a Cas9 protein and a protein having histone acetyltransferase activity, and methods of using said systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 15/549,842, filed Aug. 9, 2017, which is the U.S. national stageentry, under 35 U.S.C. § 371, of International Application NumberPCT/US2016/017221, filed Feb. 9, 2016, which claims priority to U.S.Provisional Application No. 62/113,569, filed Feb. 9, 2015, the entirecontents of each of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under Federal Grant No.1R01DA036865 awarded by the National Institutes of Health. TheGovernment has certain rights to this invention.

SEQUENCE LISTING

The instant application includes a Sequence Listing which has beensubmitted electronically in ASCII format is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 16, 2016, isnamed 028193-9190-W000 SL .txt and is 363,858 bytes in size.

TECHNICAL FIELD

The present disclosure is directed to CRISPR/Cas9-based gene activationsystems and methods of using said systems.

BACKGROUND

The Human Genome Project was funded and pursued based on the premisethat the sequencing of the human genome would reveal the genetic basisfor complex diseases that have a strong inheritable component, includingcardiovascular disease, neurodegenerative conditions, and metabolicdiseases such as diabetes. It was believed that this information wouldlead to new drug targets for these widespread diseases. However,thousands of genome-wide association studies (GWAS) have shown that thegenetic variation associated with these complex diseases does not occurwithin genes, but rather in intergenic regulatory regions that controlthe levels of particular genes. Similarly, approximately 20% ofMendelian disorders do not have a detectable coding mutation, suggestingthat the causal mutation is in a gene regulatory element. Importantly,it is very difficult to assign functional roles to these regulatoryelements as they often are located in distant locations from theirtarget genes. Moreover, many genes and regulatory elements fall intoeach positive hit of each GWAS study. In fact, follow-up projects to theHuman Genome Project, such as the NIH-funded Encyclopedia of DNAElements (ENCODE) and the Roadmap Epigenomics Project, have identifiedmillions of putative regulatory elements across the human genome formany human cell types and tissues.

A primary challenge of functional genomics is to develop technologiesthat directly and precisely manipulate genome function at individualloci. Projects such as ENCODE and the Roadmap Epigenomics Project haveidentified millions of epigenetic marks across the human genome for manyhuman cell types and tissues. Studying the function of those marks,however, has been largely limited to statistical associations with geneexpression. Technologies for targeted direct manipulation of theseepigenetic properties are necessary to transform such association-basedfindings into mechanistic principles of gene regulation. Such advanceshave the potential to benefit human health, as they could lead to genetherapies that modify the epigenetic code at targeted regions of thegenome, strategies for regenerative medicine and disease modeling basedon the epigenetic reprogramming of cell lineage specification, and theengineering of epigenome-specific drug screening platforms.

Manipulation of the epigenome is possible by treating cells with smallmolecule drugs, such as inhibitors of histone deacetylases or DNAmethyltransferases, or differentiating cells into specific lineages.However, small molecule-based methods globally alter the epigenome andtranscriptome, and are not suitable for targeting individual loci.Epigenome editing technologies, including the fusion ofepigenome-modifying enzymes to programmable DNA-binding proteins such aszinc finger proteins and transcription activator-like effectors (TALEs),have been effective at achieving targeted DNA methylation, DNAhydroxymethylation, and histone demethylation, methylation, anddeacetylation.

Fused to activation domains, such as oligomers of the herpes simplexviral protein 16 (VP16), dCas9 can function as a synthetictranscriptional regulator. However, limitations in the use of dCas9activators remain, including the need for multiple activation domains orcombinations of gRNAs to achieve high levels of gene induction bysynergistic effects between activation domains. The conventionalactivator domains used in these engineered transcriptional factors, suchas the VP16 tetramer VP64, function as a scaffold for recruitingmultiple components of the preinitiation complex and do not have directenzymatic function to specifically modulate the chromatin state. Thisindirect method of epigenetic remodeling does not allow for testing therole of specific epigenetic marks and may not be as potent as the directprogramming of epigenetic states. There remains a need for the abilityto target direct manipulation of epigenetic properties.

SUMMARY

The present invention is directed to a fusion protein comprising twoheterologous polypeptide domains, wherein the first polypeptide domaincomprises a Clustered Regularly Interspaced Short Palindromic Repeatsassociated (Cas) protein and the second polypeptide domain

The present invention is directed to a DNA targeting system comprisingthe fusion protein, described above, and at least one guide RNA (gRNA).

The present invention is directed to a method of activating geneexpression of a target gene in a cell, the method comprising contactingthe cell with a polynucleotide encoding a DNA targeting system, whereinthe DNA targeting system comprises the fusion protein, described above,and at least one guide RNA (gRNA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show that dCas9^(p300 Core) fusion protein activatestranscription of endogenous genes from proximal promoter regions. FIG.1A shows a schematic of dCas9 fusion proteins dCas9^(V64),dCas9^(FL p300), and dCas9^(p300 Core) . Streptococcus pyogenes dCas9contains nuclease inactivating mutations D10A and H840A. The D1399catalytic residue in the p300 HAT domain is indicated. FIG. 1B showsWestern blot showing expression levels of dCas9 fusion proteins andGAPDH in co-transfected cells (full blot shown in FIG. 7C). FIG. 1Cshows relative mRNA expression of IL1RN, MYOD, and OCT4, determined byqRT-PCR, by the indicated dCas9 fusion protein co-transfected with fourgRNAs targeted to each promoter region (Tukey-test, *P-value <0.05, n=3independent experiments each, error bars: s.e.m.). Numbers above barsindicate mean expression. FLAG, epitope tag; NLS, nuclear localizationsignal; HA, hemagglutinin epitope tag; CH, cysteine-histidine-richregion; Bd, bromodomain; HAT, histone acetyltransferase domain.

FIGS. 2A-2C show that dCas9^(p300 Core) fusion protein activatestranscription of endogenous genes from distal enhancer regions. FIG. 2Ashows relative MYOD mRNA production in cells co-transfected with a poolof gRNAs targeted to either the proximal or distal regulatory regionsand dCas9^(VP64) or dCas9^(p300 Core); promoter data from FIG. 1C(Tukey-test, * P-value <0.05 compared to mock-transfected cells, Tukeytest †P-value <0.05 between dCas9^(300 Core) and dCas9^(VP64), n=3independent experiments, error bars: s.e.m.). The human MYOD locus isschematically depicted with corresponding gRNA locations in red. CE,MyoD core enhancer; DRR, MyoD distal regulatory region. FIG. 2B showsrelative OCT4mRNA production in cells co-transfected with a pool ofgRNAs targeted to the proximal and distal regulatory regions anddCas9^(VP64) or dCas9^(p300 Core); promoter data from FIG. 1C(Tukey-test, *P-value <0.05 compared to mock-transfected cells, Tukeytest †P-value <0.05 between dCas9^(300 Core) and dCas9^(VP64), n=3independent experiments, error bars: s.e.m.). The human OCT4 locus isschematically depicted with corresponding gRNA locations in red. DE,Oct4 distal enhancer; PE, Oct4 proximal enhancer. FIG. 2C shows thehuman β-globin locus is schematically depicted with approximatelocations of the hypersensitive site 2 (HS2) enhancer region anddownstream genes (HBE, HBG, HBD, and HBB). Corresponding HS2 gRNAlocations are shown in red. Relative mRNA production from distal genesin cells co-transfected with four gRNAs targeted to the HS2 enhancer andthe indicated dCas9 proteins. Note logarithmic y-axis and dashed redline indicating background expression (Tukey test among conditions foreach β-globin gene, †P-value <0.05, n=3 independent experiments, errorbars: s.e.m.). n.s., not significant.

FIGS. 3A-3C show that dCas9^(p300 Core) targeted transcriptionalactivation is specific and robust. FIGS. 3A-3C show MA plots generatedfrom DEseq2 analysis of genome-wide RNA-seq data from HEK293T cellstransiently co-transfected with dCas9^(VP64) (FIG. 3A) dCas9^(p300 Core)(FIG. 3B) or dCas9^(300 Core (D1399Y)) (FIG. 3C) and four IL1RNpromoter-targeting gRNAs compared to HEK293T cells transientlyco-transfected with dCas9 and four IL1RN promoter-targeting gRNAs. mRNAscorresponding to IL1RN isoforms are shown in blue and circled in each ofFIGS. 3A-3C. Red labeled points in FIGS. 3B and 3C correspond tooff-target transcripts significantly enriched after multiple hypothesistesting (KDR, (FDR=1.4×10⁻³); FAM49A, (FDR=0.04); p300, (FDR=1.7×10⁻⁴)in FIG. 3B; and p300, (FDR=4.4×10⁻¹⁰) in FIG. 3C.

FIGS. 4A-4D show that dCas9^(p300 Core) fusion protein acetylateschromatin at a targeted enhancer and corresponding downstream genes.FIG. 4A shows the region encompassing the human β-globin locus onchromosome 11 (5,304,000-5,268,000; GRCh37/hg19 assembly) is shown. HS2gRNA target locations are indicated in red and ChIP-qPCR ampliconregions are depicted in black with corresponding green numbers.ENCODE/Broad Institute H3K27ac enrichment signal in K562 cells is shownfor comparison. Magnified insets for the HS2 enhancer, HBE, and HBG1/2promoter regions are displayed below. FIGS. 4B-4D show H3K27ac ChIP-qPCRenrichment (relative to dCas9; red dotted line) at the HS2 enhancer, HBEpromoter, and HBG1/2 promoters in cells co-transfected with four gRNAstargeted to the HS2 enhancer and the indicated dCas9 fusion protein. HBGChIP amplicons 1 and 2 amplify redundant sequences at the HBG1 and HBG2promoters (denoted by ‡). Tukey test among conditions for each ChIP-qPCRregion, *P-value <0.05 (n=3 independent experiments, error bars:s.e.m.).

FIGS. 5A-5G show that dCas9^(300 Core) fusion protein activatestranscription of endogenous genes from regulatory regions with a singlegRNA. Relative IL1RN (FIG. 5A), MYOD (FIG. 5B) or OCT4 (FIG. 5C) mRNAproduced from cells co-transfected with dCas9^(300 Core) or dCas9^(VP64)and gRNAs targeting respective promoters (n=3 independent experiments,error bars: s.e.m.). Relative MYOD (FIG. 5D) or OCT4 (FIG. 5E) mRNAproduced from cells co-transfected with dCas9^(300 Core) and indicatedgRNAs targeting the indicated MYOD or OCT4 enhancers (n=3 independentexperiments, error bars: s.e.m.). DRR, MYOD distal regulatory region;CE, MYOD core enhancer; PE, OCT4 proximal enhancer; DE, OCT4 distalenhancer. (Tukey test between dCas9^(300 Core) and single OCT4 DE gRNAscompared to mock-transfected cells, *P-value <0.05, Tukey test amongdCas9^(300 Core) and OCT4 DE gRNAs compared to Al1, †P-value <0.05).Relative HBE (FIG. 5F) or HBG (FIG. 5G) mRNA production in cellsco-transfected with dCas9^(300 Core) and the indicated gRNAs targeted tothe HS2 enhancer (Tukey test between dCas9^(300 Core) and single HS2gRNAs compared to mock-transfected cells, *P-value <0.05, Tukey testamong dCas9^(300 Core) and HS2 single gRNAs compared to Al1, †P<0.05,n=3 independent experiments, error bars: s.e.m.). HS2, β-globin locuscontrol region hypersensitive site 2; n.s., not significant using Tukeytest.

FIGS. 6A-6H show that the ^(p300 Core) can be targeted to genomic lociby diverse programmable DNA-binding proteins. FIG. 6A shows schematic ofthe Neisseria meningitidis (Nm) dCas9 fusion proteins Nm-dCas9^(VP64)and Nm-dCas9^(300 Core) Neisseria meningitidis dCas9 containsnuclease-inactivating mutations D16A, D587A, H588A, and N611A. FIG. 6Bshows relative HBE mRNA in cells co-transfected with five individual orpooled (A-E) Nm gRNAs targeted to the HBE promoter and Nm-dCas9^(VP64)or Nm-dCas9^(300 Core) FIGS. 6C-6D Relative HBE (FIG. 6C) or HBG (FIG.6D) mRNA in cells co-transfected with five individual or pooled (A-E) NmgRNAs targeted to the HS2 enhancer and Nm-dCas9^(VP64) orNm-dCas9^(300 Core) FIG. 6E shows schematic of TALEs with domainscontaining IL1RN-targeted repeat variable diresidues (Repeat Domain).FIG. 6F shows relative IL1RNmRNA in cells transfected with individual orpooled (A-D) IL1RN TALE^(V64) or IL1RN TALE^(p300 Core) encodingplasmids. FIG. 6G shows schematic of ZF fusion proteins with zinc fingerhelices 1-6 (F1-F6) targeting the ICAM1 promoter. FIG. 6H shows relativeICAM1 mRNA in cells transfected with ICAM1 ZF^(VP64) or ICAM1ZF^(300 Core) Tukey-test, *P-value <0.05 compared to mock-transfectedcontrol, n=3 independent experiments each, error bars: s.e.m. NLS,nuclear localization signal; HA, hemagglutinin tag; Bd, bromodomain; CH,cysteine-histidine-rich region; HAT, histone acetyltransferase domain.

FIGS. 7A-7C show dCas9^(p300Core) mutant fusion protein activities. FIG.7A shows schematic depiction of the WT dCas9^(p300Core) fusion proteinand ^(p300 Core) mutant derivatives. Relative locations of mutated aminoacids are displayed as yellow bars within the ^(p300 Core) effectordomain. FIG. 7B shows dCas9^(300 Core) variants were transientlyco-transfected with four IL1RN promoter gRNAs and were screened forhyperactivity¹ (amino acid 1645/1646 RR/EE and C1204R mutations) orhypoactivity (denoted by ‡) via mRNA production from the IL1RN locus(top panel, n=2 independent experiments, error bars: s.e.m.).Experiments were performed in duplicate with one well used for RNAisolation and the other for western blotting to validate expression(bottom panels). The nitrocellulose membrane was cut and incubated withα-FLAG primary antibody (top, Sigma-Aldrich cat. #F7425) or α-GAPDH(bottom, Cell Signaling Technology cat. #14C10) then α-Rabbit HRPsecondary antibody (Sigma-Aldrich cat. #A6154). FIG. 7C shows fullmembranes from western blot shown in main text (FIG. 1B). Thenitrocellulose membrane was cut and incubated with α-FLAG primaryantibody (top, Sigma-Aldrich cat. #F7425) or α-GAPDH (bottom, CellSignaling Technology cat. #14C10) then α-Rabbit HRP secondary antibody(Sigma-Aldrich cat. #A6154). Membrane was imaged for the indicateddurations after careful re-alignment of trimmed pieces.

FIG. 8 shows target gene activation is unaffected by overexpression ofsynthetic dCas9 fusion proteins.

FIGS. 9A-9E show a comparison of Sp. dCas9 and Nm. dCas9 gene inductionfrom the HS2 enhancer with individual and pooled gRNAs. FIG. 9A showsschematic display of the human β-globin locus including Streptococcuspyogenes dCas9 (Sp. dCas9) and Neisseria meningitidis dCas9 (Nm. dCas9)gRNA locations at the HS2 enhancer. Layered transcription profilesscaled to a vertical viewing range of 8 from nine ENCODE cell lines(GM12878, H1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK, and NHLF) isshown in addition to ENCODE p300 binding peaks in K562, A549 (EtOH0.02), HeLA-S3, and SKN_SH_RA cell lines. An ENCODE HEK293T DNasehypersensitive site (HEK293T DHS) is shown in the HS2 Enhancer inset.FIGS. 9B-9E shows relative transcriptional induction of HBE, HBG, HBD,and HBD transcripts from single and pooled Sp. dCas9 gRNAs (A-D) orsingle and pooled Nm. dCas9 gRNAs (A-E) in response to co-transfectionwith Sp. dCas9^(p300 Core) or Nm. dCas9^(p300 Core) respectively. gRNAsare tiled for each dCas9 ortholog corresponding to their location inGRCh37/hg19. Gray dashed line indicates background expression level intransiently co-transfected HEK293T cells. Note shared logarithmic scaleamong FIGS. 9B-9E. Numbers above bars in FIGS. 9B-9E indicate meanexpression (n=at least 3 independent experiments, error bars: s.e.m.).

FIG. 10 shows that dCas9^(V64) and dCas9^(p300 Core) induce H3K27acenrichment at IL1RN gRNA-targeted chromatin.

FIGS. 11A-11C show a direct comparison of VP64 and ^(p300 Core) effectordomains between TALE and dCas9 programmable DNA binding proteins. FIG.11A shows the GRCh37/hg19 region encompassing the IL1RN transcriptionstart site is shown schematically along with IL1RN TALE binding sitesand dCas9 IL1RN gRNA target sites. FIG. 11B shows direct comparison ofIL1RN activation in HEK293T cells when transfected with individual orpooled (A-D) IL1RN TALE^(V64) fusion proteins or when co-transfectedwith dCas9^(V64) and individual or pooled (A-D) IL1RN-targeting gRNAs.FIG. 11C shows direct comparison of IL1RN activation in HEK293T cellswhen transfected with individual or pooled (A-D)IL1RN TALE^(p300 Core)fusion proteins or when co-transfected with dCas9^(p300 Core) andindividual or pooled (A-D) IL1RN-targeting gRNAs. Note sharedlogarithmic scale between FIG. 11B and FIG. 11C. Numbers above bars inFIGS. 11B and 11C indicate mean values. Tukey test, *P-value <0.05, n=atleast 3 independent experiments, error bars: s.e.m.

FIGS. 12A-12B show TALE and ZF fusion protein expression. FIG. 12A showsWestern blotting was carried out on cells transiently transfected withindividual or pooled IL1RN TALE proteins. Nitrocellulose membranes werecut and probed with α-HA primary antibody (1:1000 dilution in TBST+5%Milk, top, Covance cat. #MMS-101P) or α-GAPDH (bottom, Cell SignalingTechnology cat. #14C10) then α-Mouse HRP (Santa Cruz, sc-2005) orα-Rabbit HRP (Sigma-Aldrich cat. #A6154) secondary antibody,respectively. FIG. 12B shows Western blotting was carried out on cellstransiently transfected with ICAM1 ZF-effector proteins andnitrocellulose membranes were cut and probed with α-FLAG primaryantibody (top, Sigma-Aldrich cat. #F7425) or α-GAPDH (bottom, CellSignaling Technology cat. #14C10) then α-Rabbit HRP secondary antibody(Sigma-Aldrich cat. #A6154). Red asterisk indicates non-specific band.

FIGS. 13A-13B show that dCas9^(p300 Core) and dCas9^(V64) do not displaysynergy in transactivation. FIG. 13A shows dCas9^(p300 Core) wasco-transfected at a 1:1 mass ratio to PL-SIN-EF1α-EGFP³ (GFP), dCas9, ordCas9^(V64) with four IL1RN promoter gRNAs as indicated (n=2 independentexperiments, error bars: s.e.m.). FIG. 13B shows dCas9^(p300 Core) wasco-transfected at a 1:1 mass ratio to GFP, dCas9, or dCas9^(V64) withfour MYOD promoter gRNAs as indicated (n=2 independent experiments,error bars: s.e.m.). No significant differences were observed usingTukey's test (n.s.).

FIGS. 14A-14D show the underlying chromatin context of dCas9^(p300 Core)target loci. FIGS. 14A-14D show indicated loci along with associatedStreptococcus pyogenes gRNAs used in this study at corresponding genomiclocations in GRCh37/hg19. ENCODE HEK293T DNase hypersensitivityenrichment is shown (note changes in scale) along with regions ofsignificant DNase hypersensitivity in HEK293T cells (“DHS”). In additionENCODE master DNase clusters across 125 cell types are shown. LayeredENCODE H3K27ac and H3K4me3 enrichment across seven cell lines (GM12878,H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) is also displayed and scaledto a vertical viewing range of 50 and 150 respectively. Endogenous p300binding profiles are also indicated for each locus and respective cellline.

FIG. 14E shows an overview of the information provided in FIGS. 14A-14D.

FIGS. 15A-15J show the amino acid sequences of dCas9 constructs.

FIG. 16 shows the amino acid sequences of ICAM1 Zinc Finger¹⁰ effectors.

FIG. 17 shows gRNA design and screening.

FIG. 18 shows gRNA combination activation.

FIG. 19 shows Pax7 guide screening in 293 Ts.

FIG. 20 shows that gRNA19 localizes to a DHS.

FIG. 21 shows the relative quantity of FGF1A mRNA in 293 Ts with orwithout dCas9^(p300 Core).

FIG. 22 shows expression levels of FGF1B and FGF1C in 293 Ts withdCas9^(p300 Core), dCas9^(V64), or dCas9 alone.

FIG. 23 shows expression levels of FGF1A, FGF1B, and FGF1C in 293 Tswith dcas9^(p300) core dCas9^(V64), or dCas9 alone.

DETAILED DESCRIPTION

Disclosed herein are CRISPR/Cas9-based gene activation systems andmethods of using said systems. The systems provide an easilyprogrammable approach to facilitate robust control of the epigenome anddownstream gene expression. The CRISPR/Cas9-based gene activation systemincludes a CRISPR/Cas9-based acetyltransferase, which is a fusionprotein of a Cas9 protein and a protein having histone acetyltransferaseactivity, such as the catalytic histone acetyltransferase (HAT) coredomain of the human E1A-associated protein p300. The Cas9 protein maynot have nuclease activity. An example of a Cas9 protein where thenuclease activity has been abolished is dCas9. Recruitment of theacetyltransferase function by dCas9 and a gRNA to the genomic targetsite allow direct modulation of epigenetic structure, and thus providean effective means of gene activation.

The disclosed CRISPR/Cas9-based acetyltransferase catalyzes acetylationof histone H3 lysine 27 at its target sites, leading to robusttranscriptional activation of target genes from promoters and bothproximal and distal enhancers. As disclosed herein, gene activation bythese targeted acetyltransferases is highly specific across the genome.The CRISPR/Cas9-based acetyltransferase, which can be targeted to anysite in the genome, is uniquely capable of activating distal regulatoryelements. In contrast to conventional dCas9-based activators, theCRISPR/Cas9-based acetyltransferase effectively activates genes fromenhancer regions and with individual or single guide RNAs.

1. Definitions

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

“Adeno-associated virus” or “AAV” as used interchangeably herein refersto a small virus belonging to the genus Dependovirus of the Parvoviridaefamily that infects humans and some other primate species. AAV is notcurrently known to cause disease and consequently the virus causes avery mild immune response.

“Chromatin” as used herein refers to an organized complex of chromosomalDNA associated with histones.

“Cis-regulatory elements” or “CREs” as used interchangeably hereinrefers to regions of non-coding DNA which regulate the transcription ofnearby genes. CREs are found in the vicinity of the gene, or genes, theyregulate. CREs typically regulate gene transcription by functioning asbinding sites for transcription factors. Examples of CREs includepromoters and enhancers.

“Clustered Regularly Interspaced Short Palindromic Repeats” and“CRISPRs”, as used interchangeably herein refers to loci containingmultiple short direct repeats that are found in the genomes ofapproximately 40% of sequenced bacteria and 90% of sequenced archaea.

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered. The coding sequence may be codonoptimize.

“Complement” or “complementary” as used herein means a nucleic acid canmean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.“Complementarity” refers to a property shared between two nucleic acidsequences, such that when they are aligned antiparallel to each other,the nucleotide bases at each position will be complementary.

“Endogenous gene” as used herein refers to a gene that originates fromwithin an organism, tissue, or cell. An endogenous gene is native to acell, which is in its normal genomic and chromatin context, and which isnot heterologous to the cell. Such cellular genes include, e.g., animalgenes, plant genes, bacterial genes, protozoal genes, fungal genes,mitochondrial genes, and chloroplastic genes.

“Enhancer” as used herein refers to non-coding DNA sequences containingmultiple activator and repressor binding sites. Enhancers range from 200bp to 1 kb in length and may be either proximal, 5′ upstream to thepromoter or within the first intron of the regulated gene, or distal, inintrons of neighboring genes or intergenic regions far away from thelocus. Through DNA looping, active enhancers contact the promoterdependently of the core DNA binding motif promoter specificity. 4 to 5enhancers may interact with a promoter. Similarly, enhancers mayregulate more than one gene without linkage restriction and may “skip”neighboring genes to regulate more distant ones. Transcriptionalregulation may involve elements located in a chromosome different to onewhere the promoter resides. Proximal enhancers or promoters ofneighboring genes may serve as platforms to recruit more distalelements.

“Fusion protein” as used herein refers to a chimeric protein createdthrough the joining of two or more genes that originally coded forseparate proteins. The translation of the fusion gene results in asingle polypeptide with functional properties derived from each of theoriginal proteins.

“Genetic construct” as used herein refers to the DNA or RNA moleculesthat comprise a nucleotide sequence that encodes a protein. The codingsequence includes initiation and termination signals operably linked toregulatory elements including a promoter and polyadenylation signalcapable of directing expression in the cells of the individual to whomthe nucleic acid molecule is administered. As used herein, the term“expressible form” refers to gene constructs that contain the necessaryregulatory elements operable linked to a coding sequence that encodes aprotein such that when present in the cell of the individual, the codingsequence will be expressed.

“Histone acetyltransferases” or “HATs” are used interchangeably hereinrefers to enzymes that acetylate conserved lysine amino acids on histoneproteins by transferring an acetyl group from acetyl CoA to formc-N-acetyllysine. DNA is wrapped around histones, and, by transferringan acetyl group to the histones, genes can be turned on and off. Ingeneral, histone acetylation increases gene expression as it is linkedto transcriptional activation and associated with euchromatin. Histoneacetyltransferases can also acetylate non-histone proteins, such asnuclear receptors and other transcription factors to facilitate geneexpression.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid may be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that may hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance may be accommodated withoutloss of promoter function.

“p300 protein,” “EP300,” or “E1A binding protein p300” as usedinterchangeably herein refers to the adenovirus E1A-associated cellularp300 transcriptional co-activator protein encoded by the EP300 gene.p300 is a highly conserved acetyltransferase involved in a wide range ofcellular processes. p300 functions as a histone acetyltransferase thatregulates transcription via chromatin remodeling and is involved withthe processes of cell proliferation and differentiation.

“Promoter” as used herein means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which may be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

“Target enhancer” as used herein refers to enhancer that is targeted bya gRNA and CRISPR/Cas9-based gene activation system. The target enhancermay be within the target region.

“Target gene” as used herein refers to any nucleotide sequence encodinga known or putative gene product. The target gene includes theregulatory regions, such as the promoter and enhancer regions, thetranscribed regions, which include the coding regions, and otherfunction sequence regions.

“Target region” as used herein refers to a cis-regulatory region or atrans-regulatory region of a target gene to which the guide RNA isdesigned to recruit the CRISPR/Cas9-based gene activation system tomodulate the epigenetic structure and allow the activation of geneexpression of the target gene.

“Target regulatory element” as used herein refers to a regulatoryelement that is targeted by a gRNA and CRISPR/Cas9-based gene activationsystem. The target regulatory element may be within the target region.

“Transcribed region” as used herein refers to the region of DNA that istranscribed into single-stranded RNA molecule, known as messenger RNA,resulting in the transfer of genetic information from the DNA moleculeto the messenger RNA. During transcription, RNA polymerase reads thetemplate strand in the 3′ to 5′ direction and synthesizes the RNA from5′ to 3′. The mRNA sequence is complementary to the DNA strand.

“Transcriptional Start Site” or “TSS” as used interchangeably hereinrefers to the first nucleotide of a transcribed DNA sequence where RNApolymerase begins synthesizing the RNA transcript.

“Transgene” as used herein refers to a gene or genetic materialcontaining a gene sequence that has been isolated from one organism andis introduced into a different organism. This non-native segment of DNAmay retain the ability to produce RNA or protein in the transgenicorganism, or it may alter the normal function of the transgenicorganism's genetic code. The introduction of a transgene has thepotential to change the phenotype of an organism.

“Trans-regulatory elements” as used herein refers to regions ofnon-coding DNA which regulate the transcription of genes distant fromthe gene from which they were transcribed. Trans-regulatory elements maybe on the same or different chromosome from the target gene.

“Variant” used herein with respect to a nucleic acid means (i) a portionor fragment of a referenced nucleotide sequence; (ii) the complement ofa referenced nucleotide sequence or portion thereof; (iii) a nucleicacid that is substantially identical to a referenced nucleic acid or thecomplement thereof; or (iv) a nucleic acid that hybridizes understringent conditions to the referenced nucleic acid, complement thereof,or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes may be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes may besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids may also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector may be a viral vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid. For example,the vector may encode a CRISPR/Cas9-based acetyltransferase having anamino acid sequence of SEQ ID NO: 140, 141, or 149 and/or at least onegRNA nucleotide sequence of any one of SEQ ID NOs: 23-73, 188-223, or224-254.

2. CRISPR/Cas9-Based Gene Activation System

Provided herein are CRISPR/Cas9-based gene activation systems for use inactivating gene expression of a target gene. The CRISPR/Cas9-based geneactivation system includes a fusion protein of a Cas9 protein that doesnot have nuclease activity, such as dCas9, and a histoneacetyltransferase or histone acetyltransferase effector domain. Histoneacetylation, carried out by histone acetyltransferases (HATs), plays afundamental role in regulating chromatin dynamics and transcriptionalregulation. The histone acetyltransferase protein releases DNA from itsheterochromatin state and allows for continued and robust geneexpression by the endogenous cellular machinery. The recruitment of anacetyltransferase by dCas9 to a genomic target site may directlymodulate epigenetic structure.

The CRISPR/Cas9-based gene activation system may catalyze acetylation ofhistone H3 lysine 27 at its target sites, leading to robusttranscriptional activation of target genes from promoters and proximaland distal enhancers. The CRISPR/Cas9-based gene activation system ishighly specific and may be guided to the target gene using as few as oneguide RNA. The CRISPR/Cas9-based gene activation system may activate theexpression of one gene or a family of genes by targeting enhancers atdistant locations in the genome.

a) CRISPR System

The CRISPR system is a microbial nuclease system involved in defenseagainst invading phages and plasmids that provides a form of acquiredimmunity. The CRISPR loci in microbial hosts contain a combination ofCRISPR-associated (Cas) genes as well as non-coding RNA elements capableof programming the specificity of the CRISPR-mediated nucleic acidcleavage. Short segments of foreign DNA, called spacers, areincorporated into the genome between CRISPR repeats, and serve as a‘memory’ of past exposures. Cas9 forms a complex with the 3′ end of thesingle guide RNA (“sgRNA”), and the protein-RNA pair recognizes itsgenomic target by complementary base pairing between the 5′ end of thesgRNA sequence and a predefined 20 bp DNA sequence, known as theprotospacer. This complex is directed to homologous loci of pathogen DNAvia regions encoded within the CRISPR RNA (“crRNA”), i.e., theprotospacers, and protospacer-adjacent motifs (PAMs) within the pathogengenome. The non-coding CRISPR array is transcribed and cleaved withindirect repeats into short crRNAs containing individual spacer sequences,which direct Cas nucleases to the target site (protospacer). By simplyexchanging the 20 bp recognition sequence of the expressed chimericsgRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPRspacers are used to recognize and silence exogenous genetic elements ina manner analogous to RNAi in eukaryotic organisms.

Three classes of CRISPR systems (Types I, II and III effector systems)are known. The Type II effector system carries out targeted DNAdouble-strand break in four sequential steps, using a single effectorenzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type IIIeffector systems, which require multiple distinct effectors acting as acomplex, the Type II effector system may function in alternativecontexts such as eukaryotic cells. The Type II effector system consistsof a long pre-crRNA, which is transcribed from the spacer-containingCRISPR locus, the Cas9 protein, and a tracrRNA, which is involved inpre-crRNA processing. The tracrRNAs hybridize to the repeat regionsseparating the spacers of the pre-crRNA, thus initiating dsRNA cleavageby endogenous RNase III. This cleavage is followed by a second cleavageevent within each spacer by Cas9, producing mature crRNAs that remainassociated with the tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNAcomplex.

An engineered form of the Type II effector system of Streptococcuspyogenes was shown to function in human cells for genome engineering. Inthis system, the Cas9 protein was directed to genomic target sites by asynthetically reconstituted “guide RNA” (“gRNA”, also usedinterchangeably herein as a chimeric sgRNA, which is a crRNA-tracrRNAfusion that obviates the need for RNase III and crRNA processing ingeneral.

The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches forsequences matching the crRNA to cleave. Target recognition occurs upondetection of complementarity between a “protospacer” sequence in thetarget DNA and the remaining spacer sequence in the crRNA. Cas9 mediatescleavage of target DNA if a correct protospacer-adjacent motif (PAM) isalso present at the 3′ end of the protospacer. For protospacertargeting, the sequence must be immediately followed by theprotospacer-adjacent motif (PAM), a short sequence recognized by theCas9 nuclease that is required for DNA cleavage. Different Type IIsystems have differing PAM requirements. The S. pyogenes CRISPR systemmay have the PAM sequence for this Cas9 (SpCas9) as 5′-NRG-3′, where Ris either A or G, and characterized the specificity of this system inhuman cells. A unique capability of the CRISPR/Cas9 system is thestraightforward ability to simultaneously target multiple distinctgenomic loci by co-expressing a single Cas9 protein with two or moresgRNAs. For example, the Streptococcus pyogenes Type II system naturallyprefers to use an “NGG” sequence, where “N” can be any nucleotide, butalso accepts other PAM sequences, such as “NAG” in engineered systems(Hsu et al., Nature Biotechnology (2013) doi:10.1038/nbt.2647).Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9)normally has a native PAM of NNNNGATT, but has activity across a varietyof PAMs, including a highly degenerate NNNNGNNN PAM (Esvelt et al.Nature Methods (2013) doi:10.1038/nmeth.2681).

b) Cas9

The CRISPR/Cas9-based gene activation system may include a Cas9 proteinor a Cas9 fusion protein. Cas9 protein is an endonuclease that cleavesnucleic acid and is encoded by the CRISPR loci and is involved in theType II CRISPR system. The Cas9 protein may be from any bacterial orarchaea species, such as Streptococcus pyogenes, Streptococcusthermophiles, or Neisseria meningitides. The Cas9 protein may be mutatedso that the nuclease activity is inactivated. In some embodiments, aninactivated Cas9 protein from Streptococcus pyogenes (iCas9, alsoreferred to as “dCas9”; SEQ ID NO: 1) may be used. As used herein,“iCas9” and “dCas9” both refer to a Cas9 protein that has the amino acidsubstitutions D10A and H840A and has its nuclease activity inactivated.In some embodiments, an inactivated Cas9 protein from Neisseriameningitides, such as NmCas9 having an amino acid sequence of SEQ ID NO:10, may be used.

c) Histone Acetyltransferase (HAT) Protein

The CRISPR/Cas9-based gene activation system may include a histoneacetyltransferase protein, such as a p300 protein, CREB binding protein(CBP; an analog of p300), GCN5, or PCAF, or fragment thereof. The p300protein regulates the activity of many genes in tissues throughout thebody. The p300 protein plays a role in regulating cell growth anddivision, prompting cells to mature and assume specialized functions(differentiate) and preventing the growth of cancerous tumors. The p300protein may activate transcription by connecting transcription factorswith a complex of proteins that carry out transcription in the cell'snucleus. The p300 protein also functions as a histone acetyltransferasethat regulates transcription via chromatin remodeling.

The histone acetyltransferase protein may include a human p300 proteinor a fragment thereof. The histone acetyltransferase protein may includea wild-type human p300 protein or a mutant human p300 protein, orfragments thereof. The histone acetyltransferase protein may include thecore lysine-acetyltransferase domain of the human p300 protein, i.e.,the p300 HAT Core (also known as “^(p300 Core)”). In some embodiments,the histone acetyltransferase protein includes an amino acid sequence ofSEQ ID NO: 2 or 3.

dCas9^(p300 Core)

The CRISPR/Cas9-based gene activation system may include a histoneacetylation effector domain. The histone acetylation effector domain maybe the catalytic histone acetyltransferase (HAT) core domain of thehuman E1A-associated protein p300 (also referred to herein as“^(p300 Core)”). In some embodiments, the ^(p300 Core) includes aminoacids 1048-1664 of SEQ ID NO: 2 (i.e., SEQ ID NO: 3). In someembodiments, the CRISPR/Cas9-based gene activation system includes adCas9^(p300 Core) fusion protein of SEQ ID NO: 141 or anNm-dCas9^(p300 Core) fusion protein of SEQ ID NO: 149. The ^(p300 Core)acetylates lysine 27 on histone H3 (H3K27ac) and may provide H3K27acenrichment.

The dCas9^(p300 Core) fusion protein is a potent and easily programmabletool to synthetically manipulate acetylation at targeted endogenousloci, leading to regulation of proximal and distal enhancer-regulatedgenes. The fusion of the catalytic core domain of p300 to dCas9 mayresult in substantially higher transactivation of downstream genes thanthe direct fusion of full-length p300 protein despite robust proteinexpression. The dCas9^(p300 Core) fusion protein may also exhibit anincreased transactivation capacity relative to dCas9^(V64), including inthe context of the Nm-dCas9 scaffold, especially at distal enhancerregions, at which dCas9^(V64) displayed little, if any, measurabledownstream transcriptional activity. Additionally, the dCas9^(p300 Core)displays precise and robust genome-wide transcriptional specificity.dCas9^(p300 Core) may be capable of potent transcriptional activationand co-enrichment of acetylation at promoters targeted by theepigenetically modified enhancer.

The dCas9^(p300 Core) may activate gene expression through a single gRNAthat target and bind a promoters and/or a characterized enhancer. Thistechnology also affords the ability to synthetically transactivatedistal genes from putative and known regulatory regions and simplifiestransactivation via the application of a single programmable effectorand single target site. These capabilities allow multiplexing to targetseveral promoters and/or enhancers simultaneously. The mammalian originof p300 may provide advantages over virally-derived effector domains forin vivo applications by minimizing potential immunogenicity.

d) gRNA

The CRISPR/Cas9-based gene activation system may include at least onegRNA that targets a nucleic acid sequence. The gRNA provides thetargeting of the CRISPR/Cas9-based gene activation system. The gRNA is afusion of two noncoding RNAs: a crRNA and a tracrRNA. The sgRNA maytarget any desired DNA sequence by exchanging the sequence encoding a 20bp protospacer which confers targeting specificity through complementarybase pairing with the desired DNA target. gRNA mimics the naturallyoccurring crRNA:tracrRNA duplex involved in the Type II Effector system.This duplex, which may include, for example, a 42-nucleotide crRNA and a75-nucleotide tracrRNA, acts as a guide for the Cas9.

The gRNA may target and bind a target region of a target gene. Thetarget region may be a cis-regulatory region or trans-regulatory regionof a target gene. In some embodiments, the target region is a distal orproximal cis-regulatory region of the target gene. The gRNA may targetand bind a cis-regulatory region or trans-regulatory region of a targetgene. In some embodiments, the gRNA may target and bind an enhancerregion, a promoter region, or a transcribed region of a target gene. Forexample, the gRNA may target and bind the target region is at least oneof HS2 enhancer of the human β-globin locus, distal regulatory region(DRR) of the MYOD gene, core enhancer (CE) of the MYOD gene, proximal(PE) enhancer region of the OCT4 gene, or distal (DE) enhancer region ofthe OCT4 gene. In some embodiments, the target region may be a viralpromoter, such as an HIV promoter.

The target region may include a target enhancer or a target regulatoryelement. In some embodiments, the target enhancer or target regulatoryelement controls the gene expression of several target genes. In someembodiments, the target enhancer or target regulatory element controls acell phenotype that involves the gene expression of one or more targetgenes. In some embodiments, the identity of one or more of the targetgenes is known. In some embodiments, the identity of one or more of thetarget genes is unknown. The CRISPR/Cas9-based gene activation systemallows the determination of the identity of these unknown genes that areinvolved in a cell phenotype. Examples of cell phenotypes include, butnot limited to, T-cell phenotype, cell differentiation, such ashematopoietic cell differentiation, oncogenesis, immunomodulation, cellresponse to stimuli, cell death, cell growth, drug resistance, or drugsensitivity.

In some embodiments, at least one gRNA may target and bind a targetenhancer or target regulatory element, whereby the expression of one ormore genes is activated. For example, between 1 gene and 20 genes,between 1 gene and 15 genes, between 1 gene and 10 genes, between 1 geneand 5 genes, between 2 genes and 20 genes, between 2 genes and 15 genes,between 2 genes and 10 genes, between 2 genes and 5 genes, between 5genes and 20 genes, between 5 genes and 15 genes, or between 5 genes and10 genes are activated by at least one gRNA. In some embodiments, atleast 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, atleast 5 gene, at least 6 genes, at least 7 genes, at least 8 genes, atleast 9 gene, at least 10 genes, at least 11 genes, at least 12 genes,at least 13 gene, at least 14 genes, at least 15 genes, or at least 20genes are activated by at least one gRNA.

The CRISPR/Cas9-based gene activation system may activate genes at bothproximal and distal locations relative the transcriptional start site(TSS). The CRISPR/Cas9-based gene activation system may target a regionthat is at least about 1 base pair to about 100,000 base pairs, at leastabout 100 base pairs to about 100,000 base pairs, at least about 250base pairs to about 100,000 base pairs, at least about 500 base pairs toabout 100,000 base pairs, at least about 1,000 base pairs to about100,000 base pairs, at least about 2,000 base pairs to about 100,000base pairs, at least about 5,000 base pairs to about 100,000 base pairs,at least about 10,000 base pairs to about 100,000 base pairs, at leastabout 20,000 base pairs to about 100,000 base pairs, at least about50,000 base pairs to about 100,000 base pairs, at least about 75,000base pairs to about 100,000 base pairs, at least about 1 base pair toabout 75,000 base pairs, at least about 100 base pairs to about 75,000base pairs, at least about 250 base pairs to about 75,000 base pairs, atleast about 500 base pairs to about 75,000 base pairs, at least about1,000 base pairs to about 75,000 base pairs, at least about 2,000 basepairs to about 75,000 base pairs, at least about 5,000 base pairs toabout 75,000 base pairs, at least about 10,000 base pairs to about75,000 base pairs, at least about 20,000 base pairs to about 75,000 basepairs, at least about 50,000 base pairs to about 75,000 base pairs, atleast about 1 base pair to about 50,000 base pairs, at least about 100base pairs to about 50,000 base pairs, at least about 250 base pairs toabout 50,000 base pairs, at least about 500 base pairs to about 50,000base pairs, at least about 1,000 base pairs to about 50,000 base pairs,at least about 2,000 base pairs to about 50,000 base pairs, at leastabout 5,000 base pairs to about 50,000 base pairs, at least about 10,000base pairs to about 50,000 base pairs, at least about 20,000 base pairsto about 50,000 base pairs, at least about 1 base pair to about 25,000base pairs, at least about 100 base pairs to about 25,000 base pairs, atleast about 250 base pairs to about 25,000 base pairs, at least about500 base pairs to about 25,000 base pairs, at least about 1,000 basepairs to about 25,000 base pairs, at least about 2,000 base pairs toabout 25,000 base pairs, at least about 5,000 base pairs to about 25,000base pairs, at least about 10,000 base pairs to about 25,000 base pairs,at least about 20,000 base pairs to about 25,000 base pairs, at leastabout 1 base pair to about 10,000 base pairs, at least about 100 basepairs to about 10,000 base pairs, at least about 250 base pairs to about10,000 base pairs, at least about 500 base pairs to about 10,000 basepairs, at least about 1,000 base pairs to about 10,000 base pairs, atleast about 2,000 base pairs to about 10,000 base pairs, at least about5,000 base pairs to about 10,000 base pairs, at least about 1 base pairto about 5,000 base pairs, at least about 100 base pairs to about 5,000base pairs, at least about 250 base pairs to about 5,000 base pairs, atleast about 500 base pairs to about 5,000 base pairs, at least about1,000 base pairs to about 5,000 base pairs, or at least about 2,000 basepairs to about 5,000 base pairs upstream from the TSS. TheCRISPR/Cas9-based gene activation system may target a region that is atleast about 1 base pair, at least about 100 base pairs, at least about500 base pairs, at least about 1,000 base pairs, at least about 1,250base pairs, at least about 2,000 base pairs, at least about 2,250 basepairs, at least about 2,500base pairs, at least about 5,000 base pairs,at least about 10,000 base pairs, at least about 11,000 base pairs, atleast about 20,000 base pairs, at least about 30,000 base pairs, atleast about 46,000 base pairs, at least about 50,000 base pairs, atleast about 54,000 base pairs, at least about 75,000 base pairs, or atleast about 100,000 base pairs upstream from the TSS.

The CRISPR/Cas9-based gene activation system may target a region that isat least about 1 base pair to at least about 500 base pairs, at leastabout 1 base pair to at least about 250 base pairs, at least about 1base pair to at least about 200 base pairs, at least about 1 base pairto at least about 100 base pairs, at least about 50 base pairs to atleast about 500 base pairs, at least about 50 base pairs to at leastabout 250 base pairs at least about 50 base pairs to at least about 200base pairs, at least about 50 base pairs to at least about 100 basepairs, at least about 100 base pairs to at least about 500 base pairs,at least about 100 base pairs to at least about 250 base pairs, or atleast about 100 base pairs to at least about 200 base pairs downstreamfrom the TSS. The CRISPR/Cas9-based gene activation system may target aregion that is at least about 1 base pair, at least about 2 base pairs,at least about 3 base pairs, at least about 4 base pairs, at least about5 base pairs, at least about 10 base pairs, at least about 15 basepairs, at least about 20 base pairs, at least about 25 base pairs, atleast about 30 base pairs, at least about 40 base pairs, at least about50 base pairs, at least about 60 base pairs, at least about 70 basepairs, at least about 80 base pairs, at least about 90 base pairs, atleast about 100 base pairs, at least about 110 base pairs, at leastabout 120, at least about 130, at least about 140 base pairs, at leastabout 150 base pairs, at least about 160 base pairs, at least about 170base pairs, at least about 180 base pairs, at least about 190 basepairs, at least about 200 base pairs, at least about 210 base pairs, atleast about 220, at least about 230, at least about 240 base pairs, orat least about 250 base pairs downstream from the TSS.

In some embodiments, the CRISPR/Cas9-based gene activation system maytarget and bind a target region that is on the same chromosome as thetarget gene but more than 100,000 base pairs upstream or more than 250base pairs downstream from the TSS. In some embodiments, theCRISPR/Cas9-based gene activation system may target and bind a targetregion that is on a different chromosome from the target gene.

The CRISPR/Cas9-based gene activation system may use gRNA of varyingsequences and lengths. The gRNA may comprise a complementarypolynucleotide sequence of the target DNA sequence followed by NGG. ThegRNA may comprise a “G” at the 5′ end of the complementarypolynucleotide sequence. The gRNA may comprise at least a 10 base pair,at least a 11 base pair, at least a 12 base pair, at least a 13 basepair, at least a 14 base pair, at least a 15 base pair, at least a 16base pair, at least a 17 base pair, at least a 18 base pair, at least a19 base pair, at least a 20 base pair, at least a 21 base pair, at leasta 22 base pair, at least a 23 base pair, at least a 24 base pair, atleast a 25 base pair, at least a 30 base pair, or at least a 35 basepair complementary polynucleotide sequence of the target DNA sequencefollowed by NGG. The gRNA may target at least one of the promoterregion, the enhancer region or the transcribed region of the targetgene. The gRNA may include a nucleic acid sequence of at least one ofSEQ ID NOs: 23-73, 188-223, or 224-254.

The CRISPR/Cas9-based gene activation system may include at least 1gRNA, at least 2 different gRNAs, at least 3 different gRNAs at least 4different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs,at least 7 different gRNAs, at least 8 different gRNAs, at least 9different gRNAs, or at least 10 different gRNAs. The CRISPR/Cas9-basedgene activation system may include between at least 1 gRNA to at least10 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, atleast 1 gRNA to at least 4 different gRNAs, at least 2 gRNA to at least10 different gRNAs, at least 2 gRNA to at least 8 different gRNAs, atleast 2 different gRNAs to at least 4 different gRNAs, at least 4 gRNAto at least 10 different gRNAs, or at least 4 different gRNAs to atleast 8 different gRNAs.

3. Target Genes

The CRISPR/Cas9-based gene activation system may be designed to targetand activate the expression of any target gene. The target gene may bean endogenous gene, a transgene, or a viral gene in a cell line. In someembodiments, the target region is located on a different chromosome asthe target gene. In some embodiments, the CRISPR/Cas9-based geneactivation system may include more than 1 gRNA. In some embodiments, theCRISPR/Cas9-based gene activation system may include more than 1different gRNAs. In some embodiments, the different gRNAs bind todifferent target regions. For example, the different gRNAs may bind totarget regions of different target genes and the expression of two ormore target genes are activated.

In some embodiments, the CRISPR/Cas9-based gene activation system mayactivate between about one target gene to about ten target genes, aboutone target genes to about five target genes, about one target genes toabout four target genes, about one target genes to about three targetgenes, about one target genes to about two target genes, about twotarget gene to about ten target genes, about two target genes to aboutfive target genes, about two target genes to about four target genes,about two target genes to about three target genes, about three targetgenes to about ten target genes, about three target genes to about fivetarget genes, or about three target genes to about four target genes. Insome embodiments, the CRISPR/Cas9-based gene activation system mayactivate at least one target gene, at least two target genes, at leastthree target genes, at least four target genes, at least five targetgenes, or at least ten target genes. For example, the may target thehypersensitive site 2 (HS2) enhancer region of the human β-globin locusand activate downstream genes (HBE, HBG, HBD and HBB).

In some embodiments, the CRISPR/Cas9-based gene activation systeminduces the gene expression of a target gene by at least about 1 fold,at least about 2 fold, at least about 3 fold, at least about 4 fold, atleast about 5 fold, at least about 6 fold, at least about 7 fold, atleast about 8 fold, at least about 9 fold, at least about 10 fold, atleast 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, atleast 90 fold, at least 100 fold, at least about 110 fold, at least 120fold, at least 130 fold, at least 140 fold, at least 150 fold, at least160 fold, at least 170 fold, at least 180 fold, at least 190 fold, atleast 200 fold, at least about 300 fold, at least 400 fold, at least 500fold, at least 600 fold, at least 700 fold, at least 800 fold, at least900 fold, or at least 1000 fold compared to a control level of geneexpression. A control level of gene expression of the target gene may bethe level of gene expression of the target gene in a cell that is nottreated with any CRISPR/Cas9-based gene activation system

The target gene may be a mammalian gene. For example, theCRISPR/Cas9-based gene activation system may target a mammalian gene,such as IL1RN, MYOD1, OCT4, HBE, HBG, HBD, HBB, MYOCD (Myocardin), PAX7(Paired box protein Pax-7), FGF1 (fibroblast growth factor-1) genes,such as FGF1A, FGF1B, and FGF1C. Other target genes include, but notlimited to, Atf3, Axud1, Btg2, c-Fos, c-Jun, Cxcl1, Cxcl2, Edn1, Ereg,Fos, Gadd45b, Ier2, Ier3, Ifrd1, Il1b, I16, Irf1, Junb, Lif, Nfkbia,Nfkbiz, Ptgs2, Slc25a25, Sqstm1, Tieg, Tnf, Tnfaip3, Zfp36, Birc2, Ccl2,Ccl20, Ccl7, Cebpd, Ch25h, CSF1, Cx3cl1, Cxcl10, Cxcl5, Gch, Icam 1,Ifi47, Ifngr2, Mmp10, Nfkbie, Npal1, p21, Relb, Ripk2, Rnd1, S1pr3,Stx11, Tgtp, T1r2, Tmem140, Tnfaip2, Tnfrsf6, Vcam1, 1110004C05Rik(GenBank accession number BC010291), Abca1, AI561871 (GenBank accessionnumber BI143915), AI882074 (GenBank accession number BB730912), Arts1,AW049765 (GenBank accession number BC026642.1), C3, Casp4, Cc15, Cc19,Cdsn, Enpp2, Gbp2, H2-D1, H2-K, H2-L, Ifit1, Ii, Il13ra1, Il1rl1, Lcn2,Lhfp12, LOC677168 (GenBank accession number AK019325), Mmp13, Mmp3, Mt2,Naf1, Ppicap, Prnd, Psmb10, Saa3, Serpina3g, Serpinf1, Sod3, Stat1,Tapbp, U90926 (GenBank accession number NM_020562), Ubd, A2AR (AdenosineA2A receptor), B7-H3 (also called CD276), B7-H4 (also called VTCN1),BTLA (B and T Lymphocyte Attenuator; also called CD272), CTLA-4(Cytotoxic T-Lymphocyte-Associated protein 4; also called CD152), IDO(Indoleamine 2,3-dioxygenase) KIR (Killer-cell Immunoglobulin-likeReceptor), LAG3 (Lymphocyte Activation Gene-3), PD-1 (Programmed Death 1(PD-1) receptor), TIM-3 (T-cell Immunoglobulin domain and Mucin domain3), and VISTA (V-domain Ig suppressor of T cell activation).

4. Compositions for Gene Activation

The present invention is directed to a composition for activating geneexpression of a target gene, target enhancer, or target regulatoryelement in a cell or subject. The composition may include theCRISPR/Cas9-based gene activation system, as disclosed above. Thecomposition may also include a viral delivery system. For example, theviral delivery system may include an adeno-associated virus vector or amodified lentiviral vector.

Methods of introducing a nucleic acid into a host cell are known in theart, and any known method can be used to introduce a nucleic acid (e.g.,an expression construct) into a cell. Suitable methods include, includee.g., viral or bacteriophage infection, transfection, conjugation,protoplast fusion, lipofection, electroporation, calcium phosphateprecipitation, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. Insome embodiments, the composition may be delivered by mRNA delivery andribonucleoprotein (RNP) complex delivery.

a) Constructs and Plasmids

The compositions, as described above, may comprise genetic constructsthat encodes the CRISPR/Cas9-based gene activation system, as disclosedherein. The genetic construct, such as a plasmid or expression vector,may comprise a nucleic acid that encodes the CRISPR/Cas9-based geneactivation system, such as the CRISPR/Cas9-based acetyltransferaseand/or at least one of the gRNAs. The compositions, as described above,may comprise genetic constructs that encodes the modified AAV vector anda nucleic acid sequence that encodes the CRISPR/Cas9-based geneactivation system, as disclosed herein. The genetic construct, such as aplasmid, may comprise a nucleic acid that encodes the CRISPR/Cas9-basedgene activation system. The compositions, as described above, maycomprise genetic constructs that encodes a modified lentiviral vector.The genetic construct, such as a plasmid, may comprise a nucleic acidthat encodes the CRISPR/Cas9-based acetyltransferase and at least onesgRNA. The genetic construct may be present in the cell as a functioningextrachromosomal molecule. The genetic construct may be a linearminichromosome including centromere, telomeres or plasmids or cosmids.

The genetic construct may also be part of a genome of a recombinantviral vector, including recombinant lentivirus, recombinant adenovirus,and recombinant adenovirus associated virus. The genetic construct maybe part of the genetic material in attenuated live microorganisms orrecombinant microbial vectors which live in cells. The geneticconstructs may comprise regulatory elements for gene expression of thecoding sequences of the nucleic acid. The regulatory elements may be apromoter, an enhancer, an initiation codon, a stop codon, or apolyadenylation signal.

The nucleic acid sequences may make up a genetic construct that may be avector. The vector may be capable of expressing the fusion protein, suchas the CRISPR/Cas9-based gene activation system, in the cell of amammal. The vector may be recombinant. The vector may compriseheterologous nucleic acid encoding the fusion protein, such as theCRISPR/Cas9-based gene activation system. The vector may be a plasmid.The vector may be useful for transfecting cells with nucleic acidencoding the CRISPR/Cas9-based gene activation system, which thetransformed host cell is cultured and maintained under conditionswherein expression of the CRISPR/Cas9-based gene activation system takesplace.

Coding sequences may be optimized for stability and high levels ofexpression. In some instances, codons are selected to reduce secondarystructure formation of the RNA such as that formed due to intramolecularbonding.

The vector may comprise heterologous nucleic acid encoding theCRISPR/Cas9-based gene activation system and may further comprise aninitiation codon, which may be upstream of the CRISPR/Cas9-based geneactivation system coding sequence, and a stop codon, which may bedownstream of the CRISPR/Cas9-based gene activation system codingsequence. The initiation and termination codon may be in frame with theCRISPR/Cas9-based gene activation system coding sequence. The vector mayalso comprise a promoter that is operably linked to theCRISPR/Cas9-based gene activation system coding sequence. TheCRISPR/Cas9-based gene activation system may be under thelight-inducible or chemically inducible control to enable the dynamiccontrol of gene activation in space and time. The promoter operablylinked to the CRISPR/Cas9-based gene activation system coding sequencemay be a promoter from simian virus 40 (SV40), a mouse mammary tumorvirus (MMTV) promoter, a human immunodeficiency virus (HIV) promotersuch as the bovine immunodeficiency virus (BIV) long terminal repeat(LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcomavirus (RSV) promoter. The promoter may also be a promoter from a humangene such as human ubiquitin C (hUbC), human actin, human myosin, humanhemoglobin, human muscle creatine, or human metalothionein. The promotermay also be a tissue specific promoter, such as a muscle or skinspecific promoter, natural or synthetic. Examples of such promoters aredescribed in US Patent Application Publication No. US20040175727, thecontents of which are incorporated herein in its entirety.

The vector may also comprise a polyadenylation signal, which may bedownstream of the CRISPR/Cas9-based gene activation system. Thepolyadenylation signal may be a SV40 polyadenylation signal, LTRpolyadenylation signal, bovine growth hormone (bGH) polyadenylationsignal, human growth hormone (hGH) polyadenylation signal, or humanβ-globin polyadenylation signal. The SV40 polyadenylation signal may bea polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego,Calif.).

The vector may also comprise an enhancer upstream of theCRISPR/Cas9-based gene activation system, i.e., the CRISPR/Cas9-basedacetyltransferase coding sequence or sgRNAs. The enhancer may benecessary for DNA expression. The enhancer may be human actin, humanmyosin, human hemoglobin, human muscle creatine or a viral enhancer suchas one from CMV, HA, RSV or EBV. Polynucleotide function enhancers aredescribed in U.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, thecontents of each are fully incorporated by reference. The vector mayalso comprise a mammalian origin of replication in order to maintain thevector extrachromosomally and produce multiple copies of the vector in acell. The vector may also comprise a regulatory sequence, which may bewell suited for gene expression in a mammalian or human cell into whichthe vector is administered. The vector may also comprise a reportergene, such as green fluorescent protein (“GFP”) and/or a selectablemarker, such as hygromycin (“Hygro”).

The vector may be expression vectors or systems to produce protein byroutine techniques and readily available starting materials includingSambrook et al., Molecular Cloning and Laboratory Manual, Second Ed.,Cold Spring Harbor (1989), which is incorporated fully by reference. Insome embodiments the vector may comprise the nucleic acid sequenceencoding the CRISPR/Cas9-based gene activation system, including thenucleic acid sequence encoding the CRISPR/Cas9-based acetyltransferaseand the nucleic acid sequence encoding the at least one gRNA comprisingthe nucleic acid sequence of at least one of SEQ ID NOs: 23-73, 188-223,or 224-254.

b) Combinations

The CRISPR/Cas9-based gene activation system composition may be combinedwith orthogonal dCas9s, TALEs, and zinc finger proteins to facilitatestudies of independent targeting of particular effector functions todistinct loci. In some embodiments, the CRISPR/Cas9-based geneactivation system composition may be multiplexed with variousactivators, repressors, and epigenetic modifiers to precisely controlcell phenotype or decipher complex networks of gene regulation.

5. Methods of Use

Potential applications of the CRISPR/Cas9-based gene activation systemare diverse across many areas of science and biotechnology. TheCRISPR/Cas9-based gene activation system may be used to activate geneexpression of a target gene or target a target enhancer or targetregulatory element. The CRISPR/Cas9-based gene activation system may beused to transdifferentiate a cell and/or activate genes related to celland gene therapy, genetic reprogramming, and regenerative medicine. TheCRISPR/Cas9-based gene activation system may be used to reprogram celllineage specification. Activation of endogenous genes encoding the keyregulators of cell fate, rather than forced overexpression of thesefactors, may potentially lead to more rapid, efficient, stable, orspecific methods for genetic reprogramming and transdifferentiation. TheCRISPR/Cas9-based gene activation system could provide a greaterdiversity of transcriptional activators to complement other tools formodulating mammalian gene expression. The CRISPR/Cas9-based geneactivation system may be used to compensate for genetic defects,suppress angiogenesis, inactivate oncogenes, activate silenced tumorsuppressors, regenerate tissue or reprogram genes.

6. Methods of Activating Gene Expression

The present disclosure provides a mechanism for activating theexpression of target genes based on targeting a histoneacetyltransferase to a target region via a CRISPR/Cas9-based geneactivation system, as described above. The CRISPR/Cas9-based geneactivation system may activate silenced genes. The CRISPR/Cas9-basedgene activation system target regions upstream of the TSS of the targetgene and substantially induced gene expression of the target gene. Thepolynucleotide encoding the CRISPR/Cas9-based gene activation system canalso be transfected directly to cells.

The method may include administering to a cell or subject aCRISPR/Cas9-based gene activation system, compositions ofCRISPR/Cas9-based gene activation system, or one or more polynucleotidesor vectors encoding said CRISPR/Cas9-based gene activation system, asdescribed above. The method may include administering aCRISPR/Cas9-based gene activation system, compositions ofCRISPR/Cas9-based gene activation system, or one or more polynucleotidesor vectors encoding said CRISPR/Cas9-based gene activation system, asdescribed above, to a mammalian cell or subject.

7. Pharmaceutical Compositions

The CRISPR/Cas9-based gene activation system may be in a pharmaceuticalcomposition. The pharmaceutical composition may comprise about 1 ng toabout 10 mg of DNA encoding the CRISPR/Cas9-based gene activationsystem. The pharmaceutical compositions according to the presentinvention are formulated according to the mode of administration to beused. In cases where pharmaceutical compositions are injectablepharmaceutical compositions, they are sterile, pyrogen free andparticulate free. An isotonic formulation is preferably used. Generally,additives for isotonicity may include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are preferred. Stabilizers include gelatinand albumin. In some embodiments, a vasoconstriction agent is added tothe formulation.

The pharmaceutical composition containing the CRISPR/Cas9-based geneactivation system may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient may be functionalmolecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient may be a transfection facilitatingagent, which may include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalene, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more preferably, thepoly-L-glutamate is present in the pharmaceutical composition containingthe CRISPR/Cas9-based gene activation system at a concentration lessthan 6 mg/ml. The transfection facilitating agent may also includesurface active agents such as immune-stimulating complexes (ISCOMS),Freunds incomplete adjuvant, LPS analog including monophosphoryl lipidA, muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid may also be used administered inconjunction with the genetic construct. In some embodiments, the DNAvector encoding the CRISPR/Cas9-based gene activation system may alsoinclude a transfection facilitating agent such as lipids, liposomes,including lecithin liposomes or other liposomes known in the art, as aDNA-liposome mixture (see for example WO9324640), calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents. Preferably, the transfectionfacilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid.

8. Methods of Delivery

Provided herein is a method for delivering the pharmaceuticalformulations of the CRISPR/Cas9-based gene activation system forproviding genetic constructs and/or proteins of the CRISPR/Cas9-basedgene activation system. The delivery of the CRISPR/Cas9-based geneactivation system may be the transfection or electroporation of theCRISPR/Cas9-based gene activation system as one or more nucleic acidmolecules that is expressed in the cell and delivered to the surface ofthe cell. The CRISPR/Cas9-based gene activation system protein may bedelivered to the cell. The nucleic acid molecules may be electroporatedusing BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb devices orother electroporation device. Several different buffers may be used,including BioRad electroporation solution, Sigma phosphate-bufferedsaline product #D8537 (PBS), Invitrogen OptiMEM I (OM), or AmaxaNucleofector solution V (N. V.). Transfections may include atransfection reagent, such as Lipofectamine 2000.

The vector encoding a CRISPR/Cas9-based gene activation system proteinmay be delivered to the mammal by DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposomemediated, nanoparticle facilitated, and/or recombinant vectors. Therecombinant vector may be delivered by any viral mode. The viral modemay be recombinant lentivirus, recombinant adenovirus, and/orrecombinant adeno-associated virus.

The nucleotide encoding a CRISPR/Cas9-based gene activation systemprotein may be introduced into a cell to induce gene expression of thetarget gene. For example, one or more nucleotide sequences encoding theCRISPR/Cas9-based gene activation system directed towards a target genemay be introduced into a mammalian cell. Upon delivery of theCRISPR/Cas9-based gene activation system to the cell, and thereupon thevector into the cells of the mammal, the transfected cells will expressthe CRISPR/Cas9-based gene activation system. The CRISPR/Cas9-based geneactivation system may be administered to a mammal to induce or modulategene expression of the target gene in a mammal. The mammal may be human,non-human primate, cow, pig, sheep, goat, antelope, bison, waterbuffalo, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats,or chicken, and preferably human, cow, pig, or chicken.

9. Routes of Administration

The CRISPR/Cas9-based gene activation system and compositions thereofmay be administered to a subject by different routes including orally,parenterally, sublingually, transdermally, rectally, transmucosally,topically, via inhalation, via buccal administration, intrapleurally,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intranasal intrathecal, and intraarticular orcombinations thereof. For veterinary use, the composition may beadministered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian may readily determine thedosing regimen and route of administration that is most appropriate fora particular animal. The CRISPR/Cas9-based gene activation system andcompositions thereof may be administered by traditional syringes,needleless injection devices, “microprojectile bombardment gone guns”,or other physical methods such as electroporation (“EP”), “hydrodynamicmethod”, or ultrasound. The composition may be delivered to the mammalby several technologies including DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposomemediated, nanoparticle facilitated, recombinant vectors such asrecombinant lentivirus, recombinant adenovirus, and recombinantadenovirus associated virus.

10. Cell Types

The CRISPR/Cas9-based gene activation system may be used with any typeof cell. In some embodiments, the cell is a bacterial cell, a fungalcell, an archaea cell, a plant cell or an animal cell. In someembodiments, the cell may be an ENCODE cell line, including but notlimited to, GM12878, K562, H1 human embryonic stem cells, HeLa-S3,HepG2, HUVEC, SK-N-SH, IMR90, A549, MCF7, HMEC or LHCM, CD14+, CD20+,primary heart or liver cells, differentiated H1 cells, 8988T, Adult_CD4naive, Adult_CD4_Th0, Adult_CD4 Th1, AG04449, AG04450, AG09309, AG09319,AG10803, AoAF, AoSMC, BC_Adipose_UHN00001, BC_Adrenal_Gland_H12803N,BC_Bladder_01-11002, BC_Brain_H11058N, BC_Breast_02-03015,BC_Colon_01-11002, BC_Colon_H12817N, BC_Esophagus_01-11002,BC_Esophagus_H12817N, BC_Jejunum_H12817N, BC_Kidney_01-11002,BC_Kidney_H12817N, BC_Left_Ventricle_N41, BC_Leukocyte_UHNO0204,BC_Liver_01-11002, BC_Lung_01-11002, BC_Lung_H12817N,BC_Pancreas_H12817N, BC_Penis_H12817N, BC_Pericardium_H12529N,BC_Placenta_UHNO0189, BC_Prostate_Gland_H12817N, BC_Rectum_N29,BC_Skeletal_Muscle_01-11002, BC_Skeletal_Muscle_H12817N,BC_Skin_01-11002, BC_Small_Intestine_01-11002, BC_Spleen_H12817N,BC_Stomach_01-11002, BC_Stomach_H12817N, BC_Testis_N30,BC_Uterus_BN0765, BE2_C, BG02ES, BG02ES-EBD, BJ, bone_marrow_HS27a,bone_marrow_HS5, bone_marrow_MSC, Breast_OC, Caco-2, CD20+_RO01778,CD20+_R001794, CD34+_Mobilized, CD4+_Naive_Wb11970640,CD4+_Naive_Wb78495824, Cerebellum_OC, Cerebrum frontal_OC, Chorion, CLL,CMK, Colo829, Colon_BC, Colon_OC, Cord_CD4_naive, Cord_CD4_Th0,Cord_CD4_Th1, Decidua, Dnd41, ECC-1, Endometrium_OC, Esophagus_BC,Fibrob1, Fibrob1_GM03348, FibroP, FibroP AG08395, FibroP AG08396, FibroPAG20443, Frontal cortex OC, GCB cell, Gliobla, GM04503, GM04504,GM06990, GM08714, GM10248, GM10266, GM10847, GM12801, GM12812, GM12813,GM12864, GM12865, GM12866, GM12867, GM12868, GM12869, GM12870, GM12871,GM12872, GM12873, GM12874, GM12875, GM12878-XiMat, GM12891, GM12892,GM13976, GM13977, GM15510, GM18505, GM18507, GM18526, GM18951, GM19099,GM19193, GM19238, GM19239, GM19240, GM20000, H0287, H1-neurons, H7-hESC,H9ES, H9ES-AFP−, H9ES-AFP+, H9ES-CM, H9ES-E, H9ES-EB, H9ES-EBD, HAc,HAEpiC, HA-h, HAL, HAoAF, HAoAF_6090101.11, HAoAF_6111301.9, HAoEC,HAoEC_7071706.1, HAoEC_8061102.1, HA-sp, HBMEC, HBVP, HBVSMC, HCF,HCFaa, HCH, HCH_0011308.2P, HCH_8100808.2, HCM, HConF, HCPEpiC, HCT-116,Heart_OC, Heart_STL003, HEEpiC, HEK293, HEK293T, HEK293-T-REx,Hepatocytes, HFDPC, HFDPC_0100503.2, HFDPC_0102703.3, HFF, HFF-Myc,HFL11W, HFL24W, HGF, HHSEC, HIPEpiC, HL-60, HMEpC, HMEpC_6022801.3, HMF,hMNC-CB, hMNC-CB_8072802.6, hMNC-CB_9111701.6, hMNC-PB,hMNC-PB_0022330.9, hMNC-PB_0082430.9, hMSC-AT, hMSC-AT_0102604.12,hMSC-AT_9061601.12, hMSC-BM, hMSC-BM_0050602.11, hMSC-BM_0051105.11,hMSC-UC, hMSC-UC 0052501.7, hMSC-UC_0081101.7, HMVEC-dAd, HMVEC-dB1-Ad,HMVEC-dB1-Neo, HMVEC-dLy-Ad, HMVEC-dLy-Neo, HMVEC-dNeo, HMVEC-LB1,HMVEC-LLy, HNPCEpiC, HOB, HOB_0090202.1, HOB_0091301, HPAEC, HPAEpiC,HPAF, HPC-PL, HPC-PL 0032601.13, HPC-PL_0101504.13, HPDE6-E6E7, HPdLF,HPF, HPIEpC, HPIEpC_9012801.2, HPIEpC_9041503.2, HRCEpiC, HRE, HRGEC,HRPEpiC, HSaVEC, HSaVEC_0022202.16, HSaVEC_9100101.15, HSMM, HSMM_emb,HSMM_FSHD, HSMMtube, HSMMtube_emb, HSMMtube_FSHD, HT-1080, HTR8svn,Huh-7, Huh-7.5, HVMF, HVMF_6091203.3, HVMF_6100401.3, HWP, HWP_0092205,HWP_8120201.5, iPS, iPS CWRU1, iPS_hFib2_iPS4, iPS_hFib2_iPS5,iPS_NIHi11, iPS_NIHi7, Ishikawa, Jurkat, Kidney_BC, Kidney_OC, LHCN-M2,LHSR, Liver_OC, Liver STL004, Liver STL011, LNCaP, Loucy, Lung BC, LungOC, Lymphoblastoid cell line, M059J, MCF10A-Er-Src, MCF-7, MDA-MB-231,Medullo, Medullo D341, Mel_2183, Melano, Monocytes-CD14+,Monocytes-CD14+_R001746, Monocytes-CD14+_R001826, MRT_A204, MRT_G401,MRT_TTC549, Myometr, Naive_B_cell, NB4, NH-A, NHBE, NHBE_RA, NHDF,NHDF_0060801.3, NHDF_7071701.2, NHDF-Ad, NHDF-neo, NHEK, NHEM.f_M2,NHEM.f_M2_5071302.2, NHEM.f_M2_6022001, NHEM_M2, NHEM_M2_7011001.2,NHEM_M2_7012303, NHLF, NT2-D1, Olf_neurosphere, Osteob1, ovcar-3,PANC-1, Pancreas_OC, PanIsletD, PanIslets, PBDE, PBDEFeta1, PBMC,PFSK-1, pHTE, Pons_OC, PrEC, ProgFib, Prostate, Prostate_OC,Psoas_muscle_OC, Raji, RCC_7860, RPMI-7951, RPTEC, RWPE1, SAEC, SH-SY5Y,Skeletal_Muscle_BC, SkMC, SKMC, SkMC_8121902.17, SkMC_9011302, SK-N-MC,SK-N-SH_RA, Small_intestine_OC, Spleen_OC, Stellate, Stomach_BC,T_cells_CD4+, T-47D, T98G, TBEC, Th1, Th1_Wb33676984, Th1_Wb54553204,Th17, Th2, Th2_Wb33676984, Th2_Wb54553204, Treg_Wb78495824,Treg_Wb83319432, U2OS, U87, UCH-1, Urothelia, WERI-Rb-1, and WI-38.

11. Kits

Provided herein is a kit, which may be used to activate gene expressionof a target gene. The kit comprises a composition for activating geneexpression, as described above, and instructions for using saidcomposition. Instructions included in kits may be affixed to packagingmaterial or may be included as a package insert. While the instructionsare typically written or printed materials they are not limited to such.Any medium capable of storing such instructions and communicating themto an end user is contemplated by this disclosure. Such media include,but are not limited to, electronic storage media (e.g., magnetic discs,tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.As used herein, the term “instructions” may include the address of aninternet site that provides the instructions.

The composition for activating gene expression may include a modifiedAAV vector and a nucleotide sequence encoding a CRISPR/Cas9-based geneactivation system, as described above. The CRISPR/Cas9-based geneactivation system may include CRISPR/Cas9-based acetyltransferase, asdescribed above, that specifically binds and targets a cis-regulatoryregion or trans-regulatory region of a target gene. TheCRISPR/Cas9-based acetyltransferase, as described above, may be includedin the kit to specifically bind and target a particular regulatoryregion of the target gene.

12. Examples

The foregoing may be better understood by reference to the followingexamples, which are presented for purposes of illustration and are notintended to limit the scope of the invention.

Example 1 Methods and Materials—Activator

Cell lines and transfection. HEK293T cells were procured from theAmerican Tissue Collection Center (ATCC, Manassas Va.) through the DukeUniversity Cell Culture Facility. Cells were cultured in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% FBS and 1%penicillin/streptomycin and maintained at 37° C. and 5% CO₂.Transfections were performed in 24-well plates using 375 ng ofrespective dCas9 expression vector and 125 ng of equimolar pooled orindividual gRNA expression vectors mixed with Lipofectamine 2000 (LifeTechnologies, cat. #11668019) as per manufacturer's instruction. ForChIP-qPCR experiments, HEK293T cells were transfected in 15 cm disheswith Lipofectamine 2000 and 30 μg of respective dCas9 expression vectorand 10 μg of equimolar pooled gRNA expression vectors as permanufacturer's instruction.

Plasmid Constructs.

pcDNA-dCas9^(VP64) (dCas9^(VP64); Addgene, plasmid #47107) was used(Perez-Pinera, P. et al, Nature methods 10:973-976 (2013)). An HAepitope tag was added to dCas9 (no effector) by removing the VP64effector domain from dCas9^(V64) via AscI/PacI restriction sites andusing isothermal assembly (Gibson et al. Nat. Methods 6:343-345 (2009))to include an annealed set of oligos containing the appropriate sequenceas per manufacturers instruction (NEB cat. #2611).)pcDNA-dCas9^(FLp300)(dCas9^(FLp300) was created by amplifying full-length p300 frompcDNA3.1-p300 (Addgene, plasmid #23252) (Chen et al. EMBO J.21:6539-6548 (2002)) in two separate fragments and cloning thesefragments into the dCas9^(V64) backbone via isothermal assembly. Asubstitution in the full-length p300 protein (L553M), located outside ofthe HAT Core region, was identified in dCas9^(FLp300) and in theprecursor pcDNA3.1-p300 during sequence validation.pcDNA-dCas9^(p300 Core) (dCas9^(p300 Core)) was generated by firstamplifying amino acids 1048-1664 of human p300 from cDNA and thensubcloning the resulting amplicon into pCR-Blunt (pCR-Blunt^(p300 Core))(Life Technologies cat. #K2700). An AscI site, HA-epitope tag, and aPmeI site were added by PCR amplification of the ^(p300 Core) frompCR-Blunt^(p300 Core) and subsequently this amplicon was cloned intopCR-Blunt (pCR-Blunt^(p300 Core+HA)) (Life Technologies cat. #K2700).The HA-tagged ^(p300 Core) was cloned from pCR-Blunt^(p300 Core+HA) intothe dCas9^(V64) backbone via shared AscI/PmeI restriction sites.pcDNA-dCas9^(p300 Core (D1399Y)) (dCas9^(p300 Core) (D1399Y)) wasgenerated by amplification of the p300 Core from dCas9^(p300 Core) inoverlapping fragments with primer sets including the specified nucleicacid mutations, with a subsequent round of linkage PCR and cloning intothe dCas9^(p300 Core) backbone using shared AscI/PmeI restriction sites.All PCR amplifications were carried out using Q5 high-fidelity DNApolymerase (NEB cat. #M0491). Protein sequences of all dCas9 constructsare shown in FIGS. 15A-15J.

IL1RN, MYOD, and OCT4 promoter gRNA protospacers have been describedpreviously (Perez-Pinera, P. et al, Nature methods 10:973-976 (2013);Hu, J. et al., Nucleic Acids Res 42:4375-4390 (2014)). Neisseriameningitidis dCas9^(V64) (Nm-dCas9^(V64)) was obtained from Addgene(plasmid #48676). Nm-dCas9^(p300 Core) was created by amplifying theHA-tagged p300 Core from dCas9^(p300 Core) with primers to facilitatesubcloning into the AleI/AgeI-digested Nm-dCas9^(V64) backbone usingisothermal assembly (NEB cat. #2611). IL1RN TALE^(p300 Core) TALEs weregenerated by subcloning the HA-tagged ^(p300 Core) domain fromdCas9^(p300 Core) into previously published (Perez-Pinera, P. et al,Nature methods 10:973-976 (2013)) IL1RN TALE^(VP64) constructs viashared AscI/PmeI restriction sites. IL1RN TALE target sites are shown inTable 1.

TABLE 1 IL1RN TAL effector information. SEQ Location (GRCh37/ NameTarget Site ID NO hg19 assembly) IL1RN TALE^(VP64 A) GGGCTCCTCCTTGTACT15 chr2:113875431-113875447 IL1RN TALE^(VP64 B) ACGCAGATAAGAACCAGT 16chr2:113875291-113875308 IL1RN TALE^(VP64 C) GGCATCAAGTCAGCCAT 17chr2:113875356-113875372 IL1RN TALE^(VP64 D) AGCCTGAGTCACCCTCCT 18chr2:113875321-113875338 IL1RN TALE^(p300 Core A) GGGCTCCTCCTTGTACT 19chr2:113875431-113875447 IL1RN TALE^(p300 Core B) ACGCAGATAAGAACCAGT 20chr2:113875291-113875308 IL1RN TALE^(p300 Core C) GGCATCAAGTCAGCCAT 21chr2:113875356-113875372 IL1RN TALE^(p300 Core D) AGCCTGAGTCACCCTCCT 22chr2:113875321-113875338

ICAM1 ZF^(V64) and ICAM1 ZF^(p300 Core) were constructed by subcloningthe ICAM1 ZF from pMX-CD54-31Opt-VP64⁵⁴ into dCas9^(V64) anddCas9^(p300 Core) backbones, respectively, using isothermal assembly(NEB cat. #2611). Protein sequences of ICAM1 ZF constructs are shown inFIG. 16. Transfection efficiency was routinely above 90% as assayed byco-transfection of PL-SIN-EF1α-EGFP (Addgene plasmid #21320) and gRNAempty vector in all experiments. All Streptococcus pyogenes gRNAs wereannealed and cloned into pZdonor-pSPgRNA (Addgene plasmid #47108) forexpression (Cong, L. et al., Science 339:819-823 (2013)) with slightmodifications using NEB BbsI and T4 ligase (Cat. #s R0539 and M0202).Nm-dCas9 gRNA oligos were rationally designed using published PAMrequirements (Esvelt, K. M. et al., Nature Methods 10:1116-1121 (2013)),and then cloned into pZDonor-Nm-Cas9-gRNA-hU6 (Addgene, plasmid #61366)via BbsI sites. Plasmids are available through Addgene (Table 2).

TABLE 2 Referenced plasmids in this study available at Addgene. PlasmidName Addgene Plasmid # pcDNA-dCas9^(VP64) (SEQ ID NO: 139) 47107pcDNA-dCas9-HA (SEQ ID NO: 138) 61355 pcDNA3.1-p300 23252pcDNA-dCas9^(FLp300) (SEQ ID NO: 140) 61356 pcDNA-dCas9^(p300 Core) (SEQID NO: 141) 61357 pcDNA-dCas9^(p300 Core (D1399Y)) (SEQ ID NO: 142)61358 pcDNA-dCas9^(p300 Core (1645/1646 RR/EE)) (SEQ ID NO: 143) 61359pcDNA-dCas9^(p300 Core (C1204R)) (SEQ ID NO: 144) 61361pcDNA-dCas9^(p300 Core (Y1467F)) (SEQ ID NO: 145) 61362pcDNA-dCas9^(p300 Core (1396/1397 SY/WW)) (SEQ ID NO: 146) 61363pcDNA-dCas9^(p300 Core (H1415A/E1423A/Y1424A/L1428S/Y1430A/H1434A))61364 (SEQ ID NO: 147) pZdonor-pSPgRNA 47108 pcDNA3.1-300(HAT-) 23254pcDNA3.3-Nm-dCas9^(VP64) (SEQ ID NO: 148) 48676pcDNA3.3-Nm-dCas9^(p300 Core) (SEQ ID NO: 149) 61365pZDonor-NmCas9-gRNA-hU6 61366 PL-SIN-EF1α-EGFP 21320

All gRNA protospacer targets are listed in Tables 3 and 4.

TABLE 3 gRNA information. Target Protospacer Sequence SEQ IDGenomic Location Location (5′-3′) NO (GRCh37/hg19 Assembly) IL1RNTGTACTCTCTGAGGTGCTC 23 chr2:113875442-113875460 Promoter A IL1RNACGCAGATAAGAACCAGTT 24 chr2:113875291-113875309 Promoter B IL1RNCATCAAGTCAGCCATCAGC 25 chr2:113875358-113875376 Promoter C IL1RNGAGTCACCCTCCTGGAAAC 26 chr2:113875326-113875344 Promoter D MYODCCTGGGCTCCGGGGCGTTT 27 chr11:17741056-17741074 Promoter A MYODGGCCCCTGCGGCCACCCCG 28 chr11:17740969-17740987 Promoter B MYODCTCCCTCCCTGCCCGGTAG 29 chr11:17740897-17740915 Promoter C MYODAGGTTGGAAAGGGCGTGC 30 chr11:17740837-17740855 Promoter D OCT4ACTCCACTGCACTCCAGTCT 31 chr6:31138711-31138730 Promoter A OCT4TCTGTGGGGGACCTGCACTG 32 chr6:31138643-31138662 Promoter B OCT4GGGGCGCCAGTTGTGTCTCC 33 chr6:31138613-31138632 Promoter C OCT4ACACCATTGCCACCACCATT 34 chr6:31138574-31138593 Promoter D MYOD DRR ATGTTTTCAGCTTCCAAACT 35 chr11:17736528-17736546 MYOD DRR BCATGAAGACAGCAGAAGCC 36 chr11:17736311-17736329 MYOD DRR CGGCCCACATTCCTTTCCAG 37 chr11:17736158-17736176 MYOD DRR DGGCTGGATTGGGTTTCCAG 38 chr11:17736065-17736083 MYOD CE ACAACTGAGTCCTGAGGTTT 39 chr11:17721347-17721365 MYOD CE BCTCACAGCACAGCCAGTGT 40 chr11:17721257-17721275 MYOD CE CCAGCAGCTGGTCACAAAGC 41 chr11:17721200-17721218 MYOD CE DCTTCCTATAAACTTCTGAG 42 chr11:17721139-17721157 OCT4 PE AAGTGATAAGACACCCGCTTT 43 chr6:31139524-31139543 OCT4 PE BCAGACATCTAATACCACGGT 44 chr6:31139604-31139623 OCT4 PE CAGGGAGAACGGGGCCTACCG 45 chr6:31139620-31139639 OCT4 PE DACTTCAGGTTCAAAGAAGCC 46 chr6:31139725-31139744 OCT4 PE ETTTTCCCCACCCAGGGCCTA 47 chr6:31139671-31139690 OCT4 PE FCCCTGGGTGGGGAAAACCAG 48 chr6:31139675-31139694 OCT4 DE AGGAGGAACATGCTTCGGAAC 49 chr6:31140809-31140828 OCT4 DE BGTGCCGTGATGGTTCTGTCC 50 chr6:31140864-31140883 OCT4 DE CGGTCTGCCGGAAGGTCTACA 51 chr6:31140707-31140726 OCT4 DE DTCGGCCTTTAACTGCCCAAA 52 chr6:31140757-31140776 OCT4 DE EGCATGACAAAGGTGCCGTGA 53 chr6:31140875-31140894 OCT4 DE FCCTGCCTTTTGGGCAGTTAA 54 chr6:31140764-31140783 HS2 AAATATGTCACATTCTGTCTC 55 chr11:5301800-5301819 HS2 B GGACTATGGGAGGTCACTAA56 chr11:5302108-5302127 HS2 C GAAGGTTACACAGAACCAGA 57chr11:5302033-5302052 HS2 D GCCCTGTAAGCATCCTGCTG 58chr11:5301898-5301917

TABLE 4 Genomic Location Protospacer Sequence SEQ (GRCh37/Target Location (5′-3′) ID NO hg19 Assembly) HBG Promoter ACCACTGCTAACTGAAAGAGA 59 chr11:5271570-5271589 HBG Promoter BAGCCACAGTTTCAGCGCAGT 60 chr11:5271692-5271711 HBG Promoter CCTGTTTCATCTTAGAAAAAT 61 chr11:5271793-5271812 HBG Promoter DGAATGTTCTTTGGCAGGTAC 62 chr11:5271942-5271961 HBG Promoter ECGCACATCTTATGTCTTAGA 63 chr11:5272021-5272040 HBE Promoter ACTTAAGAGAGCTAGAACTGG 64 chr11:5291618-5291637 HBE Promoter BTCCCAAAGTACAGTACCTTG 65 chr11:5291758-5291777 HBE Promoter CTCCCTAGAGAGGACAGACAG 66 chr11:5291785-5291804 HBE Promoter DTCATAGAGAAATGAAAAGAG 67 chr11:5291840-5291859 HBE Promoter EATAATATACCCTGACTCCTA 68 chr11:5292038-5292057 HS2 A AGGCCACCTGCAAGATAAAT69 chr11:5301662-5301681 HS2 B TGTTGTTATCAATTGCCATA 70chr11:5301708-5301727 HS2 C ATCCCTTCCAGCATCCTCAT 71chr11:5302187-5302206 HS2 D GTGCTTCAAAACCATTTGCT 72chr11:5302245-5302264 HS2 E GATACATGTTTTATTCTTAT 73chr11:5302306-5302325

Western Blotting.

20 μg of protein was loaded for SDS PAGE and transferred onto anitrocellulose membrane for western blots. Primary antibodies (a-FLAG;Sigma-Aldrich cat. #F7425 and α-GAPDH; Cell Signaling Technology cat.#14C10) were used at a 1:1000 dilution in TBST+5% Milk. Secondaryα-Rabbit HRP (Sigma-Aldrich cat. #A6154) was used at a 1:5000 dilutionin TBST+5% Milk. Membranes were exposed after addition of ECL (Bio-Radcat. #170-5060).

Quantitative Reverse-Transcription PCR.

RNA was isolated from transfected cells using the RNeasy Plus mini kit(Qiagen cat. #74136) and 500 ng of purified RNA was used as template forcDNA synthesis (Life Technologies, cat. #11754). Real-time PCR wasperformed using PerfeCTa SYBR Green FastMix (Quanta Biosciences, cat.#95072) and a CFX96 Real-Time PCR Detection System with a C1000 ThermalCycler (Bio-Rad). Baselines were subtracted using the baselinesubtraction curve fit analysis mode and thresholds were automaticallycalculated using the Bio-Rad CFX Manager software version 2.1. Resultsare expressed as fold change above control mock transfected cells (NoDNA) after normalization to GAPDH expression using the ΔΔCt method(Schmittgen et al., Nat. Protoc. 3:1101-1108 (2008)). All qPCR primersand conditions are listed in Table 5.

TABLE 5 Quantitative reverse transcription PCR and ChIP-qPCR primers andconditions. Forward SEQ Reverse SEQ Target Primer (5′-3′) ID NOPrimer (5′-3′) ID NO Cycling Parameters GAPDH CAATGACCCCTTCATT  74TTGATTTTGGAGGGA  75 95° C. 30 sec GACC TCTCG 95° C. 5 sec53° C. 20 sec| 45X IL1RN GGAATCCATGGAGGG  76 TGTTCTCGCTCAGGTC  7795° C. 30 sec AAGAT AGTG 95° C. 5 sec 58° C. 20 sec| 45X MYODTCCCTCTTTCACGGTC  78 AACACCCGACTGCTG  79 95° C. 30 sec TCAC TATCC95° C. 5 sec 53° C. 20 sec| 45X OCT4 CGAAAGAGAAAGCGA  80 CGTTGTGCATAGTCG 81 95° C. 30 sec ACCAGTATCGAGAAC CTGCTTGATCGC 95° C. 5 sec53° C. 20 sec| 45X HBB GCACGTGGATCCTGAG  82 ATTGGACAGCAAGAA  8395° C. 30 sec AACT AGCGAG 95° C. 5 sec 58° C. 20 sec| 45X HBDGCACGTGGATCCTGAG  84 CAGGAAACAGTCCAG  85 95° C. 30 sec AACT GATCTCA95° C. 5 sec 58° C. 20 sec| 45X HBG GCTGAGTGAACTGCAC  86 GAATTCTTTGCCGAA 87 95° C. 30 sec TGTGA ATGGA 95° C. 5 sec 58° C. 20 sec| 45X HBETCACTAGCAAGCTCTC  88 AACAACGAGGAGTCT  89 95° C. 30 sec AGGC GCCC95° C. 5 sec 62° C. 20 sec| 45X ICAM1 GCAGACAGTGACCATC  90CAATCCCTCTCGTCC  91 95° C. 30 sec TACAGCTT AGTCG 95° C. 5 sec58° C. 20 sec| 45X HS2 TGCTTGGACTATGGGA  92 GCAGGTGCTTCAAAA  9395° C. 30 sec ChIP GGTC CCATT 95° C. 5 sec Region 1 60° C. 20 sec| 45XHS2 TCAGGTGGTCAGCTTC  94 AAGCAAACCTTCTGG  95 95° C. 30 sec ChIP TCCTCTCAA 95° C. 5 sec Region 2 60° C. 20 sec| 45X HS2 CCACACAGGTGAACCC  96GGACACATGCTCACA  97 95° C. 30 sec ChIP TTTT TACGG 95° C. 5 sec Region 360° C. 20 sec| 45X HBE ATTCGATCCATGTGCC  98 CAATGCTGGAATTTG  9995° C. 30 sec ChIP TGA TGGAA 95° C. 5 sec Region 1 60° C. 20 sec| 45XHBE GGGGTGATTCCCTAGA 100 AAGCAGGACAGACA 101 95° C. 30 sec ChIP GAGGGGCAAG 95° C. 5 sec Region 2 60° C. 20 sec| 45X HBE GAGGGTCAGCAGTGA 102TGGAAAAGGAGAATG 103 95° C. 30 sec ChIP TGGAT GGAGA 95° C. 5 sec Region 360° C. 20 sec| 45X HBG1/2 TGGTCAAGTTTGCCTT 104 GGAATGACTGAATCG 10595° C. 30 sec ChIP GTCA GAACAA 95° C. 5 sec Region 1 60° C. 20 sec| 45XHBG1/2 CCTCCAGCATCTTCCA 106 GAAGCACCCTTCAGC 107 95° C. 30 sec ChIP CATTAGTTC 95° C. 5 sec Region 2 60° C. 20 sec| 45X HBG1/2 CCACAGTTTCAGCGCA108 ATCAGCCAGCACACA 109 95° C. 30 sec ChIP GTAATA CACTT 95° C. 5 secRegion 3 60° C. 20 sec| 45X IL1RN CCCTGTCAGGAGGGAC 110 GGCTCACCGGAAGCA111 95° C. 30 sec ChIP AGAT TGAAT 95° C. 5 sec Region 160° C. 20 sec| 45X IL1RN AAGCTACAAGCAGGTT 112 AATAACAGGGTCCAT 11395° C. 30 sec ChIP CGCT CCCGC 95° C. 5 sec Region 2 60° C. 20 sec| 45XIL1RN TGTTCCCTCCACCTGG 114 GGGAAAATCCAAAGC 115 95° C. 30 sec ChIP AATAAGGAT 95° C. 5 sec Region 3 60° C. 20 sec| 45X IL1RN TCCTAGGTCCCTCAAA116 GTCCCCAACGCTCTA 117 95° C. 30 sec ChIP AGCA ACAAA 95° C. 5 secRegion 4 60° C. 20 sec| 45X IL1RN GTTAGAGCGTTGGGGA 118 CACATGCAGAGAACT119 95° C. 30 sec ChIP CCTT GAGCTG 95° C. 5 sec Region 560° C. 20 sec| 45X IL1RN GTTGGGGTAAGCACG 120 TTTCCAGGAGGGTGA 12195° C. 30 sec ChIP AAGG CTCAG 95° C. 5 sec Region 6 60° C. 20 sec| 45XIL1RN TTCTCTGCATGTGACC 122 ACACACTCACAGAGG 123 95° C. 30 sec ChIP TCCCGTTGG 95° C. 5 sec Region 7 60° C. 20 sec| 45X IL1RN TGAGTCACCCTCCTGG124 CTCCTTCCAGAGCAC 125 95° C. 30 sec ChIP AAAC CTCAG 95° C. 5 secRegion 8 60° C. 20 sec| 45X IL1RN GCTGGGCTCCTCCTTG 126 GCTGCTGCCCATAAA127 95° C. 30 sec ChIP TACT GTAGC 95° C. 5 sec Region 960° C. 20 sec| 45X IL1RN GGACTGTGGCCCAGGT 128 GGCCTCATAGGACAG 12995° C. 30 sec ChIP ACT GAGGT 95° C. 5 sec Region 60° C. 20 sec| 45X 10IL1RN TTATGGGCAGCAGCTC 130 GACATTTTCCTGGAC 131 95° C. 30 sec ChIP AGTTGCTTG 95° C. 5 sec Region 60° C. 20 sec| 45X 11 IL1RN CCCTCCCCATGGCTTT132 AGCTCCATGCGCTTG 133 95° C. 30 sec ChIP AGGT ACATT 95° C. 5 secRegion 60° C. 20 sec|45X  12 IL1RN AGCGTCCAGGAAAAT 134 ATGACCCTCACACTC135 95° C. 30 sec ChIP GTCAA CAAGG 95° C. 5 sec Region60° C. 20 sec| 45X 13 Upstream GTTGGGTGCTCCAGCT 136 CCTCAAAACTCCTGG 13795° C. 30 sec β-actin TTTA ACTCG 95° C. 5 sec ChIP 60° C. 20 sec| 45XNEG CTRL

RNA-seq.

RNA-seq was performed using three replicates per experimental condition.RNA was isolated from transfected cells using the RNeasy Plus mini kit(Qiagen cat. #74136) and 1 μg of purified mRNA was used as template forcDNA synthesis and library construction using the PrepX RNA-Seq LibraryKit (Wafergen Biosystems, cat. #400039). Libraries were prepared usingthe Apollo 324 liquid handling platform, as per manufacturer'sinstruction. Indexed libraries were validated for quality and sizedistribution using the Tapestation 2200 (Agilent) and quantified by qPCRusing the KAPA Library Quantification Kit (KAPA Biosystems; KK4835)prior to multiplex pooling and sequencing at the Duke University GenomeSequencing Shared Resource facility. Libraries were pooled and then 50bp single-end reads were sequenced on a Hiseq 2500 (Illumina),de-multiplexed and then aligned to the HG19 transcriptome using Bowtie 2(Langmead et al. Nat. Methods 9:357-359 (2012)). Transcript abundancewas calculated using the SAMtools (Li et al. Bioinformatics 25:2078:2079(2009)) suite and differential expression was determined in R using theDESeq2 analysis package. Multiple hypothesis correction was performedusing the method of Benjamin and Hochberg with a FDR of <5%. RNA-seqdata is deposited in the NCBI's Gene Expression Omnibus and isaccessible through GEO Series accession number GSE66742.

ChIP-qPCR.

HEK293T cells were co-transfected with four HS2 enhancer gRNA constructsand indicated dCas9 fusion expression vectors in 15 cm plates inbiological triplicate for each condition tested. Cells were cross-linkedwith 1% Formaldehyde (final concentration; Sigma F8775-25ML) for 10 minat RT and then the reaction was stopped by the addition of glycine to afinal concentration of 125 mM. From each plate ˜2.5e7 cells were usedfor H3K27ac ChIP-enrichment. Chromatin was sheared to a median fragmentsize of 250 bp using a Bioruptor XL (Diagenode). H3K27ac enrichment wasperformed by incubation with 5 μg of Abcam ab4729 and 200 μl of sheepanti-rabbit IgG magnetic beads (Life Technologies 11203D) for 16 hrs at4° C. Cross-links were reversed via overnight incubation at 65° C. withsodium dodecyl sulfate, and DNA was purified using MinElute DNApurification columns (Qiagen). 10 ng of DNA was used for subsequent qPCRreactions using a CFX96 Real-Time PCR Detection System with a C1000Thermal Cycler (Bio-Rad). Baselines were subtracted using the baselinesubtraction curve fit analysis mode and thresholds were automaticallycalculated using the Bio-Rad CFX Manager software version 2.1. Resultsare expressed as fold change above cells co-transfected with dCas9 andfour HS2 gRNAs after normalization to β-actin enrichment using the ΔΔCtmethod (Schmittgen et al., Nat. Protoc. 3:1101-1108 (2008)). AllChIP-qPCR primers and conditions are listed in Table 5.

Example 2

A dCas9 Fusion to the p300 HAT Domain Activates Target Genes

The full-length p300 protein was fused to dCas9 (dCas9^(FLp300); FIGS.1A-1B) and assayed for its capacity for transactivation by transientco-transfection of human HEK293T cells with four gRNAs targeting theendogenous promoters of IL1RN, MYOD1 (MYOD), and POU5F1/OCT4 (OCT4)(FIG. 1C). A combination of four gRNAs targeting each promoter was used.dCas9^(FLp300) was well expressed and induced modest activation abovebackground compared to the canonical dCas9 activator fused to the VP64acidic activation domain (dCas9^(V64)) (FIGS. 1A-1C). The full-lengthp300 protein is a promiscuous acetyltransferase which interacts with amultitude of endogenous proteins, largely via its termini. In order tomitigate these interactions the contiguous region of full-length p300(2414 aa) solely required for inherent HAT activity (amino acids1048-1664), known as the p300 HAT core domain (p300 Core) was isolated.When fused to the C-terminus of dCas9 (dCas9^(p300 Core), FIGS. 1A-1B)the ^(p300 Core) domain induced high levels of transcription fromendogenous gRNA-targeted promoters (FIG. 1C). When targeted to the IL1RNand MYOD promoters, the dCas9^(p300 Core) fusion displayed significantlyhigher levels of transactivation than dCas9^(V64) (P-value 0.01924 and0.0324 respectively; FIGS. 1A-1C). These dCas9-effector fusion proteinswere expressed at similar levels (FIG. 1B, FIGS. 7A-7C) indicating thatthe observed differences were due to differences to transactivationcapacity. Additionally, no changes to target gene expression wereobserved when the effector fusions were transfected without gRNAs (FIG.8). For FIG. 8, dCas9 fusion proteins were transiently co-transfectedwith an empty gRNA vector backbone and mRNA expression of IL1RN, MYOD,and OCT4 was assayed as in the main text. Red dashed line indicatesbackground expression level from No DNA-transfected cells. n=2independent experiments, error bars: s.e.m., no significant activationwas observed for any target gene assayed.

To ensure that the ^(p300 Core) acetyltransferase activity wasresponsible for gene transactivation using the dCas9^(p300 Core) fusion,a panel of dCas9^(p300 Core) HAT-domain mutant fusion proteins wasscreened (FIGS. 7A-7C). A single inactivating amino acid substitutionwithin the HAT core domain (WT residue D1399 of full-length p300) ofdCas9^(p300 Core) (dCas9^(p300 Core (D1399Y)) (FIG. 1A) abolished thetransactivation capacity of the fusion protein (FIG. 1C), demonstratingthat intact ^(p300 Core) acetyltransferase activity was required fordCas9^(p300 Core)-mediated transactivation.

Example 3 dCas9^(p300 Core) Activates Genes from Proximal and DistalEnhancers

As p300 plays a role and is localized at endogenous enhancers, thedCas9^(p300 Core) may effectively induce transcription from distalregulatory regions with appropriately targeted gRNAs. The distalregulatory region (DRR) and core enhancer (CE) of the human MYOD locuswas targeted through co-transfection of four gRNAs targeted to eachregion and either dCas9^(V64) or dCas9^(p300 Core) (FIG. 2A). Comparedto a mock-transfected control, dCas9^(V64) did not show any inductionwhen targeted to the MYOD DRR or CE region. In contrast,dCas9^(p300 Core) induced significant transcription when targeted toeither MYOD regulatory element with corresponding gRNAs (P-value 0.0115and 0.0009 for the CE and DRR regions respectively). The upstreamproximal (PE) and distal (DE) enhancer regions of the human OCT4 genewere also targeted by co-transfection of six gRNAs and eitherdCas9^(V64) or dCas9^(p300 Core) (FIG. 2B). dCas9^(p300 Core) inducedsignificant transcription from these regions (P-value <0.0001 andP-value <0.003 for the DE and PE, respectively), whereas dCas9^(V64) wasunable to activate OCT4 above background levels when targeted to eitherthe PE or DE regions.

The well-characterized mammalian β-globin locus control region (LCR)orchestrates transcription of the downstream hemoglobin genes;hemoglobin epsilon 1 (HBE, from ˜11 kb), hemoglobin gamma 1 and 2 (HBG,from ˜30 kb), hemoglobin delta (HBD, from ˜46 kb), and hemoglobin beta(HBB, from ˜54 kb) (FIG. 2C). DNase hypersensitive sites within theβ-globin LCR serve as docking sites for transcriptional and chromatinmodifiers, including p300, which coordinate distal target geneexpression. Four gRNAs targeting the DNase hypersensitive site 2 withinthe LCR enhancer region (HS2 enhancer) were generated. These fourHS2-targeted gRNAs were co-transfected with dCas9, dCas9^(V64),dcas9^(p300 Core), or dCas9^(p300 Core (D1399Y)), and the resulting mRNAproduction from HBE, HBG, HBD, and HBB was assayed (FIG. 2C). dCas9,dCas9^(V64), and dCas9^(p300 Core (D1399Y)) were unable to transactivateany downstream genes when targeted to the HS2 enhancer. In contrast,targeting of dCas9^(p300 Core) to the HS2 enhancer led to significantexpression of the downstream HBE, HBG, and HBD genes (P-value <0.0001,0.0056, and 0.0003 between dCas9^(p300 Core) and mock-transfected cellsfor HBE, HBG, and HBD respectively). Overall, HBD and HBE appearedrelatively less responsive to synthetic ^(p300 Core)-mediated activationfrom the HS2 enhancer; a finding consistent with lower rates of generaltranscription from these two genes across several cell lines (FIGS.9A-9E).

Nevertheless, with the exception of the most distal HBB gene,dCas9^(p300 Core) exhibited a capacity to activate transcription fromdownstream genes when targeted to all characterized enhancer regionsassayed, a capability not observed for dCas9^(V64). Together, theseresults demonstrate that dCas9^(p300 Core) is a potent programmabletranscription factor that can be used to regulate gene expression from avariety of promoter-proximal and promoter-distal locations.

Example 4 Gene Activation by dCas9^(p300 Core) is Highly Specific

Recent reports indicate that dCas9 may have widespread off-targetbinding events in mammalian cells in combination with some gRNAs, whichcould potentially lead to off-target changes in gene expression. Inorder to assess the transcriptional specificity of the dCas9^(p300 Core)fusion protein, transcriptome was performed profiling by RNA-seq incells co-transfected with four IL1RN-targeted gRNAs and either dCas9,dCas9^(V64), dcas9^(p300 Core), or dCas9^(p300 Core (D1399Y)).Genome-wide transcriptional changes were compared between dCas9 with nofused effector domain and either dCas9^(V64), dcas9^(p300 Core) ordCas9^(p300 Core (D1399Y)) (FIGS. 3A-3C). While both dCas9^(V64) anddCas9^(p300 Core) upregulated all four IL1RN isoforms, only the effectsof dCas9^(p300 Core) reached genome-wide significance (FIGS. 3A-3B,Table 6; P-value 1.0×10⁻³-5.3×10′ for dCas9^(V64); P-value1.8×10⁻¹⁷-1.5×10⁻¹⁹ for dCas9^(p300 Core))

TABLE 6 Ten most enriched mRNAs for dCas9 IL1RN-targeted RNA-seqexperiments Base log2 Fold Refseq ID Gene Mean Change lfcSE stat pvaluepadj dCAS9^(VP64) + 4 IL1RN gRNAs compared to dCas9 + 4 IL1RN gRNAs 1NM_173842   IL1RN (transcript variant 1) 14.764 0.529 0.152 3.480.000494857 0.99992134 2 NM_173843   IL1RN (transcript variant 4) 13.6060.517 0.149 3.47 0.000530109 0.99992134 3 NR_073102   ZNF551 21.5050.505 0.159 3.17 0.00152863  0.99992134 4 NM_000577   IL1RN (transcriptvariant 3) 14.890 0.497 0.152 3.28 0.001039353 0.99992134 5 NM_001077441BCLAF1(transcript variant 3) 437.814 0.482 0.153 3.14 0.0016659250.99992134 6 NM_173841   IL1RN (transcript variant 2) 13.711 0.448 0.15 3.00 0.002716294 0.99992134 7 NM_001268   RCBTB2 46.265 0.440 0.167 2.640.008335513 0.99992134 8 NM_000922   PDE3B 143.947 0.439 0.167 2.630.008471891 0.99992134 9 NM_001077440 BCLAF1 (transcript variant 2)463.743 0.439 0.156 2.82 0.004790762 0.99992134 10 NM_014739   BCLAF1(transcript variant 1) 474.598 0.432 0.158 2.74 0.006232218 0.99992134dCAS9^(p300 Core) + 4 IL1RN gRNAs compared to dCas9 + 4 IL1RN gRNAs 1NM_173843 IL1RN (transcript variant 4) 45.517 1.548 0.171 9.04 1.52E−195.24E−15 2 NM_173841 IL1RN (transcript variant 2) 40.690 1.457 0.1718.50 1.83E−17 3.16E−13 3 NM_173842 IL1RN (transcript variant 1) 39.5681.448 0.171 8.45 2.88E−17 3.30E−13 4 NM_000577 IL1RN (transcript variant3) 41.821 1.437 0.171 8.39 4.88E−17 4.20E−13 5 NM_001429 p300 928.4350.955 0.171 5.57 2.50E−08 0.000171838 6 NM_002253 KDR 17.477 0.842 0.1635.17 2.36E−07 0.00135472  7 NM_030797 FAM49A 21.286 0.736 0.166 4.448.91E−06 0.043823927 8 NM_012074 DPF3 17.111 0.609 0.164 3.720.000202676 0.871938986 9 NM_031476 CRISPLD2 25.148 0.569 0.167 3.410.000653132 0.999954424 10 NM_007365 PADI2 99.012 0.554 0.162 3.410.000641145 0.999954424 dCAS9^(p300 Core (D1399Y)) + 4 IL1RN gRNAscompared to dCas9 + 4 IL1RN gRNAs 1 NM_001429   p300 935.659 1.234 0.1986.24 4.36E-10 1.49E-05 2 NM_001270493 SREK1 (transcript variant 4)30.118 0.651 0.203 3.20 0.001388089 0.999938051 3 NM_001079802 FKTN(transcript variant 1) 148.558 0.546 0.203 2.69 0.007212168 0.9999380514 NM_000922   PDE3B 140.122 0.535 0.201 2.66 0.007805491 0.999938051 5NM_206937   LIG4 (transcript variant 2) 30.589 0.521 0.203 2.560.010513626 0.999938051 6 NM_001136116 ZNF879 18.421 0.520 0.201 2.590.009600802 0.999938051 7 NM_018374   TMEM106B (transcript 280.758 0.5160.196 2.64 0.008329592 0.999938051 variant 1) 8 NM_019863   F8(transcript variant 2) 8.048 0.515 0.178 2.89 0.003827553 0.999938051 9NM_001193349 MEF2C (transcript variant 5) 18.934 0.510 0.202 2.530.011492452 0.999938051 10 NM_183245   INVS (transcript variant 2)38.545 0.497 0.203 2.45 0.014125973 0.999938051

In contrast, dCas9^(p300 Core (D1399Y)) did not significantly induce anyIL1RN expression (FIG. 3C; P-value >0.5 for all 4 IL1RN isoforms).Comparative analysis to dCas9 revealed limited dCas9^(300 Core)off-target gene induction, with only two transcripts inducedsignificantly above background at a false discovery rate (FDR)<5%: KDR(FDR=1.4×10⁻³); and FAM49A (FDR=0.04) (FIG. 3B, Table 6). Increasedexpression of p300 mRNA was observed in cells transfected withdCas9^(p300 Core) and dCas9^(p300 Core (D1399Y)). This finding is mostlikely explained by RNA-seq reads mapping to mRNA from the transientlytransfected p300 core fusion domains. Thus the dCas9^(p300 Core) fusiondisplayed high genome-wide targeted transcriptional specificity androbust gene induction of all four targeted IL1RN isoforms.

Example 5 dCas9^(p300 Core) Acetylates H3K27 at Enhancers and Promoters

Activity of regulatory elements correlates with covalent histonemodifications such as acetylation and methylation. Of those histonemodifications, acetylation of lysine 27 on histone H3 (H3K27ac) is oneof the most widely documented indicators of enhancer activity.Acetylation of H3K27 is catalyzed by p300 and is also correlated withendogenous p300 binding profiles. Therefore H3K27ac enrichment was usedas a measurement of relative dCas9^(p300 Core)-mediated acetylation atthe genomic target site. To quantify targeted H3K27 acetylation bydCas9^(p300 Core) chromatin immuno-precipitation was performed with ananti-H3K27ac antibody followed by quantitative PCR (ChIP-qPCR) inHEK293T cells co-transfected with four HS2 enhancer-targeted gRNAs andeither dCas9, dCas9^(V64), dCas9^(p300 Core) ordCas9^(p300 Core (D1399Y)) (FIGS. 4A-4D). Three amplicons were analyzedat or around the target site in the HS2 enhancer or within the promoterregions of the HBE and HBG genes (FIG. 4A). Notably, H3K27ac is enrichedin each of these regions in the human K562 erythroid cell line that hasa high level of globin gene expression (FIG. 4A). Significant H3K27acenrichment was observed at the HS2 enhancer target locus compared totreatment with dCas9 in both the dCas9^(V64) (P-value 0.0056 for ChIPRegion land P-value 0.0029 for ChIP Region 3) and dCas9^(p300 Core)(P-value 0.0013 for ChIP Region land P-value 0.0069 for ChIP Region 3)co-transfected samples (FIG. 4B).

A similar trend of H3K27ac enrichment was also observed when targetingthe IL1RN promoter with dCas9^(V64) or dCas9^(p300 Core) (FIG. 10). FIG.10 shows the IL1RN locus on GRCh37/hg19 along with IL1RN gRNA targetsites. In addition, layered ENCODE H3K27ac enrichment from seven celllines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) is indicatedwith the vertical range setting set to 50. Tiled IL1RNChIP qPCRamplicons (1-13) are also shown in corresponding locations onGRCh37/hg19. H3K27ac enrichment for dCas9^(V64) and dCas9^(p300 Core)co-transfected with four IL1RN-targeted gRNAs and normalized to dCas9co-transfected with four IL1RN gRNAs is indicated for each ChIP qPCRlocus assayed. 5ng of ChIP-prepared DNA was used for each reaction (n=3independent experiments, error bars: s.e.m.).

In contrast to these increases in H3K27ac at the target sites by bothdCas9^(V64) or dCas9^(p300 Core) robust enrichment in H3K27ac at theHS2-regulated HBE and HBG promoters was observed only withdCas9^(p300 Core) treatment (FIGS. 4C-4D). Together these resultsdemonstrate that dCas9^(p300 Core) uniquely catalyzes H3K27ac enrichmentat gRNA-targeted loci and at enhancer-targeted distal promoters.Therefore the acetylation established by dCas9^(p300 Core) at HS2 maycatalyze enhancer activity in a manner distinct from direct recruitmentof preinitiation complex components by dCas9^(V64), as indicated by thedistal activation of the HBE, HBG, and HBD genes from the HS2 enhancerby dCas9^(p300 Core) but not by dCas9^(V64) (FIG. 2C, FIGS. 9A-9E).

Example 6 dCas9^(p300 Core) Activates Genes with a Single gRNA

Robust transactivation using dCas9-effector fusion proteins currentlyrelies upon the application of multiple gRNAs, multiple effectordomains, or both. Transcriptional activation could be simplified withthe use of single gRNAs in tandem with a single dCas9-effector fusion.This would also facilitate multiplexing distinct target genes and theincorporation of additional functionalities into the system. Thetransactivation potential of dCas9^(p300 Core) with single gRNAs wascompared to that of dCas9^(p300 Core) with four pooled gRNAs targetingthe IL1RN, MYOD and OCT4 promoters (FIGS. 5A-5B). Substantial activationwas observed upon co-transfection of the dCas9^(p300 Core) and a singlegRNA at each promoter tested. For the IL1RN and MYOD promoters, therewas no significant difference between the pooled gRNAs and the bestindividual gRNA (FIGS. 5A-5B; IL1RN gRNA “C”, P-value 0.78; MYOD gRNA“D”, P-value 0.26). Although activation of the OCT4 promoter producedadditive effects when four gRNAs were pooled with dCas9^(p300 Core) themost potent single gRNA (gRNA “D”) induced a statistically comparableamount of gene expression to that observed upon co-transfection ofdCas9^(V64) with an equimolar pool of all four promoter gRNAs (P-value0.73; FIG. 5C). Compared to dCas9^(p300 Core) levels of gene activationwith dCas9^(V64) and single gRNAs were substantially lower. Also, incontrast to dCas9^(p300 Core), dCas9^(V64) demonstrated synergisticeffects with combinations of gRNAs in every case (FIGS. 5A-5C).

Based on the transactivation ability of dCas9^(p300 Core) at enhancerregions and with single gRNAs at promoter regions, it was hypothesizedthat dCas9^(p300 Core) might also be able to transactivate enhancers viaa single targeted gRNA. The MYOD (DRR and CE), OCT4 (PE and DE), and HS2enhancer regions were tested with equimolar concentrations of pools orsingle gRNAs (FIGS. 5D-5G). For both MYOD enhancer regions,co-transfection of dCas9^(p300 Core) and a single enhancer-targetinggRNA was sufficient to activate gene expression to levels similar tocells co-transfected with dCas9^(p300 Core) and the four pooled enhancergRNAs (FIG. 5D). Similarly, OCT4 gene expression was activated from thePE via dCas9^(p300 Core) localization with a single gRNA to similarlevels as dCas9^(p300 Core) localized with a pool of six PE-targetedgRNAs (FIG. 5E). dCas9^(p300 Core)_mediated induction of OCT4 from theDE (FIG. 5E) and HBE and HBG genes from the HS2 enhancer (FIGS. 5F-5G)showed increased expression with the pooled gRNAs relative to singlegRNAs. Nevertheless, there was activation of target gene expressionabove control for several single gRNAs at these enhancers (FIGS. 5E-5G).

Example 7 The p300 HAT Domain is Portable to Other DNA-Binding Proteins

The dCas9/gRNA system from Streptococcus pyogenes has been widelyadopted due to its robust, versatile, and easily programmableproperties. However, several other programmable DNA-binding proteins arealso under development for various applications and may be preferablefor particular applications, including orthogonal dCas9 systems fromother species, TALEs, and zinc finger proteins. To determine if the^(p300 Core) HAT domain was portable to these other systems, fusionswere created to dCas9 from Neisseria meningitidis (Nm-dCas9), fourdifferent TALEs targeting the IL1RN promoter, and a zinc finger proteintargeting ICAM1 (FIGS. 6A-6H). Co-transfection of Nm-dCas9^(p300 Core)and five Nm-gRNAs targeted to the HBE or the HBG promoters led tosignificant gene induction compared to mock-transfected controls(P-value 0.038 and 0.0141 for HBE and HBG respectively) (FIG. 6B). Whenco-transfected with five Nm-gRNAs targeted to the HS2 enhancer,Nm-dCas9^(p300 Core) also significantly activated the distal HBE andHBG, globin genes compared to mock-transfected controls (p=0.0192 andp=0.0393, respectively)(FIGS. 6C-6D). Similar to dCas9^(p300 Core),Nm-dCas9^(p300 Core) activated gene expression from promoters and theHS2 enhancer via a single gRNA. Nm-dCas9^(VP64) displayed negligiblecapacity to transactivate HBE or HBG regardless of localization topromoter regions or to the HS2 enhancer either with single or multiplegRNAs (FIGS. 6B-6D). Transfection of the expression plasmids for acombination of four TALE^(p300 Core) fusion proteins targeted to theIL1RN promoter (IL1RN TALE^(p300 Core)) also activated downstream geneexpression, although to a lesser extent than four correspondingTALE^(V64) fusions (IL1RN TALE^(V64)) (FIGS. 6E-6F). However, single^(p300 Core) effectors were much more potent than single VP64 domainswhen fused to IL1RN TALEs. Interestingly, dCas9^(p300 Core) directed toany single binding site generated comparable IL1RN expression relativeto single or pooled IL1RN TALE effectors and direct comparisons suggestthat dCas9 may be a more robust activator than TALEs when fused to thelarger ^(p300 Core) fusion domain (FIGS. 11A-11C). The ^(p300 Core)effector domain did not display synergy with either additional gRNAs orTALEs (see FIGS. 5A-5G, 6A-6H, 9A-9E, and 11A-11C) or in combinationwith VP64 (see FIGS. 13A-13B). The underlying chromatin context of thedCas9^(p300 Core) target loci is shown in FIGS. 14A-14E.

The ZF^(p300 Core) fusion targeted to the ICAM1 promoter (ICAM1ZF^(p300 Core)) also activated its target gene relative to control andat a similar level as ZF^(VP64)(ICAM1 ZF^(V64)) (FIGS. 6G-6H). Theversatility of the ^(p300 Core) fusion with multiple targeting domainsis evidence that this is a robust approach for targeted acetylation andgene regulation. The various p300 core fusion proteins were expressedwell, as determined by western blot (FIGS. 12A-12B), but differences in^(p300 Core) activity between different fusion proteins could beattributable to binding affinity or protein folding.

Example 8 Myocardin

36 gRNAs were designed to span −2000 bp to +250 bp (coordinates relativeto TSS) region of the MYOCD gene (Table 7).

TABLE 7 Myocd gRNAs Information SEQ SEQ Coordinates Target gRNA ID IDRelative to Name Name Protospacer (N20) NO PAM NO +/- Length TSS MyocdCr1 cctggtatcaatgagaaga 152 NGG 188 - 20 -1991 -1971 Myocd Cr2gattaggacatgaacatggg 153 NGG 189 - 20 -1897 -1877 Myocd Cr3cctcttctacattaacctta 154 NGG 190 - 20 -1771 -1751 Myocd Cr4tttttgaagccagcaatcgt 155 NGG 191 - 20 -1693 -1673 Myocd Cr5cgttagtttctggaggctct 156 NGG 192 - 20 -1597 -1577 Myocd Cr6acaaattaccacgaatgtag 157 NGG 193 - 20 -1480 -1460 Myocd Cr7tggcctgggcgcctgtctat 158 NGG 194 - 20 -1395 -1375 Myocd Cr8attttgtaaataaggtcttc 159 NGG 195 - 20 -1297 -1277 Myocd Cr9agcaacaggggatggggcag 160 NGG 196 + 20 -1221 -1201 Myocd Cr10aggactcgtagtatgcaggc 161 NGG 197 + 20 -1120 -1100 Myocd Cr11ctgagccaccaactatttaa 162 NGG 198 + 20 -1005  -985 Myocd Cr12ctgagccaccaactatttaa 163 NGG 199 + 20  -945  -925 Myocd Cr13actctgggtcggttacggaa 164 NGG 200 + 20  -907  -887 Myocd Cr14gggctgggcttagcttggga 165 NGG 201 - 20  -837  -817 Myocd Cr15atagggaggggctctggagc 166 NGG 202 - 20  -798  -778 Myocd Cr16atgggaaaagatacctgagt 167 NGG 203 - 20  -751  -731 Myocd Cr17tgggagcgttgtgtcgcagc 168 NGG 204 + 20  -713  -693 Myocd Cr18tggaaaggctttcattttct 169 NGG 205 - 20  -642  -622 Myocd Cr19gtatctcgcagctccaatac 170 NGG 206 - 20  -594  -574 Myocd Cr20acgcattcccctcggtttga 171 NGG 207 - 20  -544  -524 Myocd Cr21tcggaagcttttcttctcag 172 NGG 208 + 20  -511  -491 Myocd Cr22cgaaagggcgtgcgcgcccg 173 NGG 209 - 20  -449  -429 Myocd Cr23ccggcgaaagggaagcggcc 174 NGG 210 - 20  -396  -376 Myocd Cr24ggctgcgcacgcccatcccc 175 NGG 211 + 20  -352  -332 Myocd Cr25ggggcttgcaggtggttcgc 176 NGG 212 - 20  -297  -277 Myocd Cr26cgagctaaagagcggatgcc 177 NGG 213 - 20  -246  -226 Myocd Cr27agagggcgggagcagggcca 178 NGG 214 - 20  -200  -180 Myocd Cr28aaccggctcttaactctttg 179 NGG 215 - 20  -153  -133 Myocd Cr29caggagcggcgagcggggtc 180 NGG 216 - 20  -101   -81 Myocd Cr30gggtatcagatggcaaagtt 181 NGG 217 + 20   -54   -34 Myocd Cr31tcataggctgccggcgattg 182 NGG 218 - 20     0    20 Myocd Cr32gaggttggccaggagcagcg 183 NGG 219 - 20    47    67 Myocd Cr33aattagccccgcacggcgag 184 NGG 220 + 20   100   120 Myocd Cr34tcccctgggtaggagtacag 185 NGG 221 - 20   157   177 Myocd Cr35ggttgttagctgcggtcagc 186 NGG 222 + 20   203   223 Myocd Cr36ggtggagaacagggggcgcc 187 NGG 223 + 20   246   266

The gRNAs were cloned into a spCas9 gRNA expression vector containinghU6 promoter and BbsI restriction site. The gRNAs were transientlyco-transfected with dCas9^(p300 Core) into HEK293T cells. The resultingmRNA production for myocardin was assayed in samples harvested threedays post-transfection (FIG. 17). Combinations of Cr32, Cr13, Cr30,Cr28, Cr31, and Cr34 were analyzed with dCas9^(p300 Core) (Table 8; FIG.18).

TABLE 8 Condition Cr32 Cr13 Cr30 Cr28 Cr31 Cr34 1 X X X X X X 2 X X X X3 X X X X 4 X X X X 5 X X X X 6 X X X X 7 X X X X 8 X X X X 9 X X X X 10X X X X 11 X X X X X 12 X X X X X 13 X X X X X 14 X X X X X 15 X X X X X16 X X X X X

Example 9 Pax7

gRNAs were designed to span the region surrounding the PAX7 gene (Table9). The gRNAs were cloned into a spCas9 gRNA expression vectorcontaining hU6 promoter and BbsI restriction site. The gRNAs weretransiently co-transfected with dCas9^(p300 Core) or dCas9^(VP64) intoHEK293T cells. The resulting mRNA production for Pax7 was assayed insamples harvested three days post-transfection (FIG. 19). The gRNA19(“g19”) was used in further experiments and shown to localize to a DNasehypersensitive site (DHS) (FIG. 20).

TABLE 9 Pax7 gRNAs TSS Target Target SEQ ID position Strand nameOligo in sense strand NO  138 AS JK12 GGGGGCGCGAGTGATCAGCT 224   27 SJK16 CCCGGGTCTCCTAGGGGACG 225  +95 S JK17 TGGTCCGGAGAAAGAAGGCG 226 +187S JK18 GTCTCCGGGCTCGGAAACTT 227 +223 S JK19 AGCGCCAGAGCGCGAGAGCG 228+273 S JK20 CGATTCCGGCCGCGTTCCCC 229 +335 AS JK21 GTTGTGCGGGCTGATGCGCC230

Example 10 FGF1

gRNAs were designed for the FGF1A, FGF1B, and FGF1C genes (Tables 10 and11). The 25 nM of gRNAs were transiently co-transfected withdCas9^(p300 Core) or dCas9^(V64) into HEK293T cells. The resulting mRNAproduction for FGF1 expression was determined (FIGS. 21-23). In FIG. 23,the number of stable cell-lines transfected with the lentivirus vectorwas 2, except for FGF1A where n=1.

TABLE 10 gRNA Gene Type Name 1 FGF1A F_7sk 1FGF1AF_7sk 2 FGF1A F_h12FGF1AF_h1 3 FGF1A F_hU6 3FGF1AF_hU6 4 FGF1A F_mU6 4FGF1AF_mU6 1 FGF1AR_7sk 1FGF1AR_7sk 2 FGF1A R_h1 2FGF1AR_h1 3 FGF1A R_hU6 3FGF1AR_hU6 4FGF1A R_mU6 4FGF1AR_mU6 1 FGF1B F_7sk 1FGF1BF_7sk 2 FGF1B F_h12FGF1BF_h1 3 FGF1B F_hU6 3FGF1BF_hU6 4 FGF1B F_mU6 4FGF1BF_mU6 1 FGF1BR_7sk 1FGF1BR_7sk 2 FGF1B R_h1 2FGF1BR_h1 3 FGF1B R_hU6 3FGF1BR_hU6 4FGF1B R_mU6 4FGF1BR_mU6 1 FGF1C F_7sk 1FGF1CF_7sk 2 FGF1C F_h12FGF1CF_h1 3 FGF1C F_hU6 3FGF1CF_hU6 4 FGF1C F_mU6 4FGF1CF_mU6 1 FGF1CR_7sk 1FGF1CR_7sk 2 FGF1C R_h1 2FGF1CR_h1 3 FGF1C R_hU6 3FGF1CR_hU6 4FGF1C R_mU6 4FGF1CR_mU6

TABLE 11 FGF1 gRNAs Information SEQ SEQ ID 1st ID 2nd gRNAFinal Sequence NO addition Sequence NO addition 1 CCTCGTGTGTTCCTGGGC 231CCTCG TGTGTTCCTGGGCCTG 255 CTGCTGC CTGC 2 TCCCATAAACAGGATTC 232 TCCCATAAACAGGATTCTGCT 256 TGCTCAGA CAGA 3 CACCGGCCAGATGACAG 233 CACCGGCCAGATGACAGAAC 257 AACAGAAA AGAAA 4 TTGTTTGAAAATGCCATT 234 TTGTTTGAAAATGCCATTTGTA 258 TGTAGGGCT GGGCT 1 AAACGCAGCAGGCCCAG 235 AAACGCAGCAGGCCCAGGA 259 C GAACACAC ACACA 2 AAACTCTGAGCAGAATC 236 AAACTCTGAGCAGAATCCT 260 T CTGTTTAT GTTTA 3 AAACTTTCTGTTCTGTCA 237 AAACTTTCTGTTCTGTCATC 261 C TCTGGCC TGGC 4 AAACAGCCCTACAAATG 238 AAACAGCCCTACAAATGGC 262 CAA GCATTTTCAA ATTTT 1 CCTCGtctgcttctgccgaacctca 239CCTCG tctgcttctgccgaacctca 263 2 TCCCAcctaaagagcttgtaggccg 240 TCCCAcctaaagagcttgtaggccg 264 3 CACCGagagctggctacccgtccct 241 CACCGagagctggctacccgtccct 265 4 TTGTTTGcggtccttgtttatcagta 242 TTGTTTGcggtccttgtttatcagtag 266 g 1 AAACtgaggttcggcagaagcaga 243 AAACtgaggttcggcagaagcaga 267 C C 2 AAACcggcctacaagctctttaggT 244 AAACcggcctacaagctctttagg 268 T 3 AAACagggacgggtagccagctct 245 AAACagggacgggtagccagctct 269 C C 4 AAACctactgataaacaaggaccgC 246 AAACctactgataaacaaggaccg 270 CAA AA 1 CCTCGGAGCTGGCTACC 247 CCTCGGAGCTGGCTACCCGT 271 CGTCCCTA CCCTA 2 TCCCACTTTGGCTGGGTT 248 TCCCACTTTGGCTGGGTTTAA 272 TAAACCA ACCA 3 CACCGGTCAGCTCAGGG 249 CACCGGTCAGCTCAGGGTTTT 273 TTTTGGTA GGTA 4 TTGTTTGGAGTTAGCTCC 250 TTGTTTGGAGTTAGCTCCCCGA 274 CCGACCCAG CCCAG 1 AAACTAGGGACGGGTAG 251 AAACTAGGGACGGGTAGCC 275 C CCAGCTCC AGCTC 2 AAACTGGTTTAAACCCA 252 AAACTGGTTTAAACCCAGCC 276 T GCCAAAGT AAAG 3 AAACTACCAAAACCCTG 253 AAACTACCAAAACCCTGAG 277 C AGCTGACC CTGAC 4 AAACCTGGGTCGGGGAG 254 AAACCTGGGTCGGGGAGCT 278 CAA CTAACTCCAA AACTC

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A fusion protein comprising two heterologous polypeptidedomains, wherein the first polypeptide domain comprises a ClusteredRegularly Interspaced Short Palindromic Repeats associated (Cas) proteinand the second polypeptide domain comprises a peptide having histoneacetyltransferase activity.

Clause 2. The fusion protein of clause 1, wherein the fusion proteinactivates transcription of a target gene.

Clause 3. The fusion protein of clause 1 or 2, wherein the Cas proteincomprises Cas9.

Clause 4. The fusion protein of clause 3, wherein the Cas9 comprises atleast one amino acid mutation which knocks out nuclease activity ofCas9.

Clause 5. The fusion protein of clause 4, wherein the Cas proteincomprises SEQ ID NO: 1 or SEQ ID NO: 10.

Clause 6. The fusion protein of any one of clauses 1-5, wherein thesecond polypeptide domain comprises a histone acetyltransferase effectordomain.

Clause 7. The fusion protein of clause 6, wherein the histoneacetyltransferase effector domain is a p300 histone acetyltransferaseeffector domain.

Clause 8. The fusion protein of any one of clauses 1-7, wherein thesecond polypeptide domain comprises SEQ ID NO: 2 or SEQ ID NO: 3.

Clause 9. The fusion protein of any one of clauses 1-8, wherein thefirst polypeptide domain comprises SEQ ID NO: 1 or SEQ ID NO: 10 and thesecond polypeptide domain comprises SEQ ID NO: 2 or SEQ ID NO. 3.

Clause 10. The fusion protein of any one of clauses 1-9, wherein thefirst polypeptide domain comprises SEQ ID NO: 1 and the secondpolypeptide domain comprises SEQ ID NO. 3, or the first polypeptidedomain comprises SEQ ID NO: 10 and the second polypeptide domaincomprises SEQ ID NO. 3.

Clause 11. The fusion protein of any one of clauses 1-10, furthercomprising a linker connecting the first polypeptide domain to thesecond polypeptide domain.

Clause 12. The fusion protein of any one of clauses 1-11, wherein thefusion protein comprises an amino acid sequence of SEQ ID NO: 140, 141,or 149.

Clause 13. A DNA targeting system comprising the fusion protein of anyone of clauses 1-12 and at least one guide RNA (gRNA).

Clause 14. The DNA targeting system of clause 13, wherein the at leastone gRNA comprises a 12-22 base pair complementary polynucleotidesequence of the target DNA sequence followed by a protospacer-adjacentmotif.

Clause 15. The DNA targeting system of clause 13 or 14, wherein the atleast one gRNA targets a target region, the target region comprises atarget enhancer, target regulatory element, a cis-regulatory region of atarget gene, or a trans-regulatory region of a target gene.

Clause 16. The DNA targeting system of clause 15, wherein the targetregion is a distal or proximal cis-regulatory region of the target gene.

Clause 17. The DNA targeting system of clause 15 or 16, wherein thetarget region is an enhancer region or a promoter region of the targetgene.

Clause 18. The DNA targeting system of any one of clauses 15-17, whereinthe target gene is an endogenous gene or a transgene.

Clause 19. The DNA targeting system of clause 15, wherein the targetregion comprises a target enhancer or a target regulatory element.

Clause 20. The DNA targeting system of clause 19, wherein the targetenhancer or target regulatory element control the gene expression ofmore than one target gene.

Clause 21. The DNA targeting system of any one of clauses 15-20, whereinthe DNA targeting system comprises between one and ten different gRNAs.

Clause 22. The DNA targeting system of any one of clauses 15-21, whereinthe DNA targeting system comprises one gRNA.

Clause 23. The DNA targeting system of any one of clauses 15-22, whereinthe target region is located on the same chromosome as the target gene.

Clause 24. The DNA targeting system of clause 23, wherein the targetregion is located about 1 base pair to about 100,000 base pairs upstreamof a transcription start site of the target gene.

Clause 25. The DNA targeting system of clause 24, wherein the targetregion is located about 1000 base pairs to about 50,000 base pairsupstream of the transcription start site of the target gene.

Clause 26. The DNA targeting system of any one of clauses 15-22, whereinthe target region is located on a different chromosome as the targetgene.

Clause 27. The DNA targeting system of any one of clauses 15-28, whereinthe different gRNAs bind to different target regions.

Clause 28. The DNA targeting system of clause 27, wherein the differentgRNAs bind to target regions of different target genes.

Clause 29. The DNA targeting system of clause 27, wherein the expressionof two or more target genes are activated.

Clause 30. The DNA targeting system of any one of clauses 15-29, whereinthe target gene is selected from the group consisting of IL1RN, MYOD1,OCT4, HBE, HBG, HBD, HBB, MYOCD, PAX7, FGF1A, FGF1B, and FGF1C.

Clause 31. The DNA targeting system of clause 30, wherein the targetregion is at least one of HS2 enhancer of the human β-globin locus,distal regulatory region (DRR) of the MYOD gene, core enhancer (CE) ofthe MYOD gene, proximal (PE) enhancer region of the OCT4 gene, or distal(DE) enhancer region of the OCT4 gene.

Clause 32. The DNA targeting system of any one of clauses 13-31, whereinthe gRNA comprises at least one of SEQ ID NOs: 23-73, 188-223, or224-254.

Clause 33. An isolated polynucleotide encoding the fusion protein of anyone of clauses 1-12 or the DNA targeting system of any one of clauses13-32.

Clause 34. A vector comprising the isolated polynucleotide of clause 33.

Clause 35. A cell comprising the isolated polynucleotide of clause 33 orthe vector of clause 34.

Clause 36. A kit comprising the fusion protein of any one of clauses1-12, the DNA targeting system of clauses 13-32, the isolatedpolynucleotide of clause 33, the vector of clause 34, or the cell ofclause 35.

Clause 37. A method of activating gene expression of a target gene in acell, the method comprising contacting the cell with the fusion proteinof any one of clauses 1-12, the DNA targeting system of clauses 13-32,the isolated polynucleotide of clause 33, or the vector of clause 34.

Clause 38. A method of activating gene expression of a target gene in acell, the method comprising contacting the cell with a polynucleotideencoding a DNA targeting system, wherein the DNA targeting systemcomprises the fusion protein of any one of clauses 1-12 and at least oneguide RNA (gRNA).

Clause 39. The method of clause 38, wherein the at least one gRNAcomprises a 12-22 base pair complementary polynucleotide sequence of thetarget DNA sequence followed by a protospacer-adjacent motif.

Clause 40. The method of clause 38 or 39, wherein the at least one gRNAtargets a target region, the target region is a cis-regulatory region ora trans-regulatory region of a target gene.

Clause 41. The method of clause 40, wherein the target region is adistal or proximal cis-regulatory region of the target gene.

Clause 42. The method of clause 40 or 41, wherein the target region isan enhancer region or a promoter region of the target gene.

Clause 43. The method of clause 40-42, wherein the target gene is anendogenous gene or a transgene.

Clause 44. The method of clause 43, wherein the DNA targeting systemcomprises between one and ten different gRNAs.

Clause 45. The method of clause 43, wherein the DNA targeting systemcomprises one gRNA.

Clause 46. The method of clause 40-45, wherein the target region islocated on the same chromosome as the target gene.

Clause 47. The method of clause 46, wherein the target region is locatedabout 1 base pair to about 100,000 base pairs upstream of atranscription start site of the target gene.

Clause 48. The method of clause 46, wherein the target region is locatedabout 1000 base pairs to about 50,000 base pairs upstream of thetranscription start site of the target gene.

Clause 49. The method of clause 40-45, wherein the target region islocated on a different chromosome as the target gene.

Clause 50. The method of clause 40-45, wherein the different gRNAs bindto different target regions.

Clause 51. The method of clause 50, wherein the different gRNAs bind totarget regions of different target genes.

Clause 52. The method of clause 51, wherein the expression of two ormore target genes are activated.

Clause 53. The method of clause 40-52, wherein the target gene isselected from the group consisting of IL1RN, MYOD1, OCT4, HBE, HBG, HBD,HBB, MYOCD, PAX7, FGF1A, FGF1B, and FGF1C.

Clause 54. The method of clause 53, wherein the target region is atleast one of HS2 enhancer of the human β-globin locus, distal regulatoryregion (DRR) of the MYOD gene, core enhancer (CE) of the MYOD gene,proximal (PE) enhancer region of the OCT4 gene, or distal (DE) enhancerregion of the OCT4 gene.

Clause 55. The method of clause 37-54, wherein the gRNA comprises atleast one of SEQ ID NOs: 23-73, 188-223, or 224-254.

Clause 56. The method of any one of clauses 37-55, wherein the DNAtargeting system is delivered to the cell virally or non-virally.

Clause 57. The method of any one of clauses 37-56, wherein the cell is amammalian cell.

Appendix-SequencesStreptococcus pyogenes Cas 9 (with D10A, H849A) (SEQ ID NO: 1)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDHuman p300 (with L553M mutation) (SEQ ID NO: 2)MAENVVEPGPPSAKRPKLSSPALSASASDGTDFGSLFDLEHDLPDELINSTELGLTNGGDINQLQTSLGMVQDAASKHKQLSELLRSGSSPNLNMGVGGPGQVMASQAQQSSPGLGLINSMVKSPMTQAGLTSPNMGMGTSGPNQGPTQSTGMMNSPVNQPAMGMNTGMNAGMNPGMLAAGNGQGIMPNQVMNGSIGAGRGRQNMQYPNPGMGSAGNLLTEPLQQGSPQMGGQTGLRGPQPLKMGMMNNPNPYGSPYTQNPGQQIGASGLGLQIQTKTVLSNNLSPFAMDKKAVPGGGMPNMGQQPAPQVQQPGLVTPVAQGMGSGAHTADPEKRKLIQQQLVLLLHAHKCQRREQANGEVRQCNLPHCRTMKNVLNHMTHCQSGKSCQVAHCASSRQIISHWKNCTRHDCPVCLPLKNAGDKRNQQPILTGAPVGLGNPSSLGVGQQSAPNLSTVSQIDPSSIERAYAALGLPYQVNQMPTQPQVQAKNQQNQQPGQSPQGMRPMSNMSASPMGVNGGVGVQTPSLLSDSMLHSAINSQNPMMSENASVPSMGPMPTAAQPSTTGIRKQWHEDITQDLRNHLVHKLVQAIFPTPDPAALKDRRMENLVAYARKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRLQKQNMLPNAAGMVPVSMNPGPNMGQPQPGMTSNGPLPDPSMIRGSVPNQMMPRITPQSGLNQFGQMSMAQPPIVPRQTPPLQHHGQLAQPGALNPPMGYGPRMQQPSNQGQFLPQTQFPSQGMNVTNIPLAPSSGQAPVSQAQMSSSSCPVNSPIMPPGSQGSHIHCPQLPQPALHQNSPSPVPSRTPTPHHTPPSIGAQQPPATTIPAPVPTPPAMPPGPQSQALHPPPRQTPTPPTTQLPQQVQPSLPAAPSADQPQQQPRSQQSTAASVPTPTAPLLPPQPATPLSQPAVSIEGQVSNPPSTSSTEVNSQAIAEKQPSQEVKMEAKMEVDQPEPADTQPEDISESKVEDCKMESTETEERSTELKTEIKEEEDQPSTSATQSSPAPGQSKKKIFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPOTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDRFVYTCNECKHHVETRWHCTVCEDYDLCITCYNTKNHDHKMEKLGLGLDDESNNQQAAATQSPGDSRRLSIQRCIQSLVHACQCRNANCSLPSCQKMKRVVQHTKGCKRKTNGGCPICKQLIALCCYHAKHCQENKCPVPFCLNIKQKLRQQQLQHRLQQAQMLRRRMASMQRTGVVGQQQGLPSPTPATPTTPTGQQPTTPQTPQPTSQPQPTPPNSMPPYLPRTQAAGPVSQGKAAGQVTPPTPPQTAQPPLPGPPPAAVEMAMQIQRAAETQRQMAHVQIFQRPIQHQMPPMTPMAPMGMNPPPMTRGPSGHLEPGMGPTGMQQQPPWSQGGLPQPQQLQSGMPRPAMMSVAQHGQPLNMAPQPGLGQVGISPLKPGTVSQQALQNLLRTLRSPSSPLQQQQVLSILHANPQLLAAFIKQRAAKYANSNPQPIPGQPGMPQGQPGLQPPTMPGQQGVHSNPAMQNMNPMQAGVQRAGLPQQQPQQQLQPPMGGMSPQAQQMNIVINRNTMPSQFRDILRRQQMMQQQQQQGAGPGIGPGMANRNQFQQPQGVGYPPQQQQRMQHFIMQQMQQGNMGQIGQLPQALGAEAGASLQAYQQRLLQQQMGSPVQPNPMSPQQHMLPNQAQSPHLQGQQIPNSLSNQVRSPQPVPSPRPQSQPPHSSPSPRMQPQPSPHHVSPQTSSPHPGLVAAQANPMEQGHFASPDQNSMLSQLASNPGMANLHGASATDLGLSTDNSDLNSNLSQSTLDIHp300 Core Effector (aa 1048-1664 of SEQ ID NO: 2) (SEQ ID NO: 3)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDu300 Core Effector (aa 1048-1664 of SEQ ID NO: 2 with D1399Y mutation)(SEQ ID NO: 4)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLYSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDp300 Core Effector (aa 1048-1664 of SEQ ID NO: 2 with 1645/1646 RR/EEmutations) (SEQ ID NO: 5)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLEEAQWSTMCMLVELHTQSQDp300 Core Effector (aa 1048-1664 of SEQ ID NO: 2 with C1204R mutation)(SEQ ID NO: 6)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKRFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDp300 Core Effector (aa 1048-1664 of SEQ ID NO: 2 with Y1467F mutation)(SEQ ID NO: 7)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWFKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDp300 Core Effector (aa 1048-1664 of SEQ ID NO: 2 with 1396/1397 SY/WWmutations) (SEQ ID NO: 8)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYIWWLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDp300 Core Effector (aa 1048-1664 of SEQ ID NO: 2 with H1415A, E1423A,Y1424A, L14285, Y1430A, and H1434A mutations) (SEQ ID NO: 9)IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYAEILIGYLAAVKKSGATTGAIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQDNeisseria meningitidis Cas9 (with D16A, D587A, H588A, and N611Amutations) (SEQ ID NO: 10)MAAFKPNPINYILGLAIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIAAALPFSRTWDDSFNNKVLVLGSEAQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR 3X ″Flag″ Epitope (SEQ ID NO: 11)DYKDHDGDYKDHDIDYKDDDDK Nuclear Localization Sequence (SEQ ID NO: 12)PKKKRKVG HA Epitope (SEQ ID NO: 13) YPYDVPDYASVP64 Effector (SEQ ID NO: 14)DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML

1-57. (canceled)
 58. An isolated polynucleotide encoding a fusionprotein comprising two heterologous polypeptide domains, wherein thefirst polypeptide domain comprises a Clustered Regularly InterspacedShort Palindromic Repeats associated (Cas) protein, wherein the secondpolypeptide domain comprises a p300 histone acetyltransferase effectordomain, and wherein the fusion protein comprises the polypeptidesequence of SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO:
 149. 59. Theisolated polynucleotide of claim 58, wherein the fusion proteinactivates transcription of a target gene by activating a regulatoryelement.
 60. The isolated polynucleotide of claim 59, wherein theregulatory element comprises a promoter of the target gene.
 61. A vectorcomprising the isolated polynucleotide of claim
 58. 62. A kit comprisingthe isolated polynucleotide of claim
 58. 63. A cell comprising theisolated polynucleotide of claim
 58. 64. An isolated polynucleotideencoding a fusion protein comprising two heterologous polypeptidedomains, wherein the first polypeptide domain comprises a ClusteredRegularly Interspaced Short Palindromic Repeats associated (Cas)protein, wherein the second polypeptide domain comprises a p300 histoneacetyltransferase effector domain, and wherein the first polypeptidedomain comprises the polypeptide sequence of SEQ ID NO: 10 and thesecond polypeptide domain comprises the polypeptide sequence of SEQ IDNO: 3, or the first polypeptide domain comprises the polypeptidesequence of SEQ ID NO: 1 and the second polypeptide domain comprises thepolypeptide sequence of SEQ ID NO:
 2. 65. The isolated polynucleotide ofclaim 64, wherein the fusion protein activates transcription of a targetgene by activating a regulatory element.
 66. The isolated polynucleotideof claim 65, wherein the regulatory element comprises a promoter of thetarget gene.
 67. The isolated polynucleotide of claim 64, furthercomprising a linker connecting the first polypeptide domain to thesecond polypeptide domain.
 68. A vector comprising the isolatedpolynucleotide of claim
 64. 69. A kit comprising the isolatedpolynucleotide of claim
 64. 70. A cell comprising the isolatedpolynucleotide of claim 64.